Biological production of methyl methacrylate

ABSTRACT

The invention relates to an engineered eukaryotic microorganism into which a gene encoding an acyl-CoA dehydrogenase is introduced and a method for producing methacrylic acid esters such as MMA and MMA-CoA and precursors thereof using the microorganism.

RELATED APPLICATION

The present application claims priority based on U.S. Provisional Application No. 62/245,980 (filed on Oct. 23, 2015), and the content thereof is incorporated herein by reference.

TECHNICAL FIELD

The invention relates to an engineered eukaryotic microorganism wherein genes encoding an acyl-CoA dehydrogenase are introduced into the microorganism and a method for producing methacrylic acid esters and precursors thereof using the microorganism.

BACKGROUND ART

Methyl methacrylate (MMA) is a critical raw material in the production of acrylic polymers. MMA is traditionally produced from non-sustainable, hazardous raw materials, such as acetone and hydrogen cyanide. Accordingly, there is a need in the art for MMA production methods that are sustainable and rely on non-toxic production methods.

As such a production method, methods are proposed wherein 2-hydroxyisobutyric acid and 3-hydroxyisobutyric acid to be precursors of methacrylic acids are produced from natural products such as saccharides using naturally occurring microorganisms (see Patent Literatures 1 and 2 and Non Patent Literature 1). However, these methods still rely on chemical techniques in the step for producing a methacrylic acid by dehydrating the precursor.

Additionally, there are methods proposed wherein a methacrylic acid is produced from glucose using a not naturally occurring engineered microorganism into which a plurality of enzyme genes are introduced, but these are the combination of a known enzyme reaction and a hypothetical enzyme reaction analogized therefrom and not verified (see Patent Literatures 3 to 5). Particularly, Patent Literature 5 presents examples of a wide variety of biocatalysts (hydrolase, wax ester synthase, alcohol acetyltransferase) which have typical ester-producing activity but fails to state that those biocatalysts have the synthesis activity of methacrylic acid esters.

The inventors have reported a production method of a methacrylic acid ester from methacrylyl-CoA by biocatalysts (Patent Literature 6) and a production method of a methacrylic acid ester from biomass in the presence of an alcohol by adding the action of AAT to a microorganism which has a methacrylic acid producing ability (Patent Literature 7). Additionally, the inventors have reported a method for synthesizing methacrylyl-CoA from 3-hydroxyisobutyryl-CoA using E. coli into which a dehydratase gene is introduced (Patent Literature 8).

CITATION LIST Patent Literature

-   Patent Literature 1: WO2007/110394 -   Patent Literature 2: WO2008/145737 -   Patent Literature 3: WO2009/135074 -   Patent Literature 4: WO2011/031897 -   Patent Literature 5: WO2012/135789 -   Patent Literature 6: WO2014/038214 -   Patent Literature 7: WO2014/038216 -   Patent Literature 8: WO2015/015784

Non Patent Literature

-   Non Patent Literature 1: Green Chemistry, 2012, 14, 1942-1948

SUMMARY OF INVENTION Technical Problem

It is an object of the invention to provide a novel biological production method of methacrylic acid esters such as MMA.

Solution to Problem

Hosts for industrial use such as E. coli do not produce MAA-CoA even when exogenous genes related to the biosynthesis of methacrylyl-CoA (MAA-CoA) are introduced thereinto. This is presumed due to the absence of the electron acceptor for acyl-CoA dehydrogenase, which is one of the enzymes related to the biosynthesis of MAA-CoA, in E. coli. The inventors succeeded in the biological production of MAA-CoA by introducing an exogenous acyl-CoA dehydrogenase gene into a yeast cell.

More specifically, the invention relates to the following (1) to (10).

(1) A eukaryotic microorganism into which a gene encoding an acyl-CoA dehydrogenase (isobutyryl-CoA dehydrogenase) is introduced. (2) The eukaryotic microorganism according to (1), wherein the gene encoding an acyl-CoA dehydrogenase is derived from at least one selected from genus Pseudomonas, genus Bacillus, genus Sphingobacterium, genus Comamonas, genus Brevundimonas, genus Sphingomonas, genus Ochrobactrum, genus Pedobacter, genus Paenibacillus, genus Achromobacter, genus Acinetobacter, genus Shewanella, genus Listonella, genus Agrobacterium, genus Mesorhizobium, genus Rhizobium, genus Paracoccus, genus Xanthobacter, genus Streptomyces, genus Geobacillus, genus Rhodococcus, genus Saccharomyces, genus Candida and genus Aspergillus. (3) The eukaryotic microorganism according to (1) or (2), wherein a signal sequence-added acyl-CoA dehydrogenase gene is introduced in such a way that the acyl-CoA dehydrogenase expresses a function in a mitochondrion. (4) The eukaryotic microorganism according to (3), wherein the signal sequence comprises the sequence as set forth in SEQ ID NO: 1. (5) The eukaryotic microorganism according to any one of (1) to (4), wherein the eukaryotic microorganism is a yeast. (6) The eukaryotic microorganism according to any one of (1) to (4), further comprising at least one exogenous gene selected from genes encoding branched-chain keto acid dehydrogenase, genes encoding enoyl-CoA hydratase, genes encoding hydroxyacyl-CoA hydrolase, genes encoding thioesterase, and genes encoding alcohol acyl transferase. (7) A method for producing methacrylyl-CoA from valine using the eukaryotic microorganism according to (6). (8) A method for producing 3-hydroxyisobutyryl-CoA from valine using the eukaryotic microorganism according to (6). (9) A method for producing 3-hydroxyisobutyric acid from valine using the eukaryotic microorganism according to (6). (10) A method for producing a methacrylic acid ester from valine using the eukaryotic microorganism according to (6).

Provided herein are novel methods for the biological production of MMA and MMA precursors in engineered microorganisms, wherein one or more enzymes which enable MMA or MMA precursor production is introduced into the microorganism. Herein, one or more enzyme genes including an acyl-CoA dehydrogenase gene, which enables the production of MMA or an MMA. Optionally, the one or more enzymes is targeted to the mitochondria. Mitochondrial targeting of enzymes enables the biosynthetic processes which produce MMA or MMA precursors to take advantage of the favorable energetic, enzymatic, and other characteristics of the mitochondrial niche, which greatly enhances yield. In one implementation of the invention, various enzymes involved in valine biosynthesis and degradation are engineered into a host eukaryotic microorganism wherein, through a series of reactions, they produce MMA precursors or MMA from valine, the one or more enzymes being optionally targeted to the mitochondria. The scope of the invention encompasses novel gene vectors for the transformation of hosts, novel microorganism strains expressing enzymes which enable the synthesis of MMA precursors or MMA, and MMA precursors and MMA produced by the engineered microorganisms of the invention.

Advantageous Effects of Invention

According to the invention, the biological production of methacrylic acid ester precursors such as methacrylyl-CoA (MAA-CoA) is provided. The MAA precursor is biologically converted to methacrylic acid esters such as MAA to begin with, thereby enabling the bio-integrated production of methacrylic acid esters. The method of the invention is a biological production which uses an organic raw material such as biomass and thus can produce methacrylic acid esters non-toxically without destroying environments. Additionally, methacrylic acid esters to be obtained are water-insoluble, thereby being collected and purified easily from a fermented product, whereby low-cost production of methacrylic acid esters is enabled.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an overview of the synthetic pathways for the production of MMA precursors and MMA from valine.

FIG. 2 depicts synthetic pathways and experimental results in the exemplary implementation of the invention described in Example 1. Colorimetric analysis of ACD activity. The reaction progress was monitored in real time in terms of the absorption reduction rate at 600 nm.

FIG. 3 depicts synthetic pathways and experimental results in the exemplary implementation of the invention described in Example 1. The vertical axis shows the activity of recombinant ACD (acd1) expressed in S. cerevisiae after 48-hour expression/induction in a cytoplasm (MMA) and a mitochondrion (mtMMA).

FIG. 4 depicts synthetic pathways and experimental results in the exemplary implementation of the invention described in Example 1. Spectroscopic analysis of BCKAD activity. The reaction progress was monitored in real time in terms of the absorption reduction rate at 340 nm.

FIG. 5 depicts synthetic pathways and experimental results in the exemplary implementation of the invention described in Example 1. The vertical axis shows the activities of recombinant BCKAD (bkdA1, bkdA2, IpdV, bkdB) expressed in S. cerevisiae after 48-hour expression/induction in a cytoplasm (MMA) and a mitochondrion (mtMMA).

DESCRIPTION OF EMBODIMENTS Part 1: Microorganisms Capable of Producing MMA and MMA Precursors

The invention encompasses an engineered microorganism which produces methacrylic acid esters such as methyl methacrylate (MMA) or precursors thereof such as methacrylyl-CoA (MAA-CoA) and 3-hydroxyisobutyric acid (3-HIB) by the various biosynthesis pathways. Collectively, methacrylic acid esters and precursors thereof (for example, MMA and ester thereof) will be referred to herein as “MMA end-products.” The above biosynthesis pathways comprise a number of enzymatic steps that are accomplished in microorganisms engineered to possess the appropriate set of enzymatic capabilities, as described below.

In the invention, the “methacrylic acid” (IUPAC name: 2-methyl-2-propenoic acid) includes any salts or ionized forms thereof. Examples of the salt of methacrylic acid include sodium salts, potassium salts, calcium salts and magnesium salts.

The “methacrylic acid ester” is a compound represented by formula 1. In the formula 1, R represents a linear or branched hydrocarbon group having 1 to 20 carbon atoms. The hydrocarbon group may be saturated or unsaturated, acyclic or cyclic. Linear or branched-chain unsubstituted alkyl groups, aralkyl groups or aryl groups having 1 to 10 carbon atoms are preferable. Particularly preferable are alkyl groups, benzyl groups or phenyl groups having 1 to 8 carbon atoms of methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, tert-butyl group, n-pentyl group, isopentyl group, tert-pentyl group, n-hexyl group, isohexyl group, 2-hexyl group, dimethyl butyl group, ethyl butyl group, heptyl group, octyl group and 2-ethylhexyl group.

CH₂═C(CH₃)COO—R  (Formula 1)

Examples of the “methacrylic acid ester precursor” include methacrylyl-CoA, 3-hydroxyisobutyryl-CoA, 3-hydroxyisobutyric acid and isobutyryl-CoA.

Engineered Microorganisms

The invention encompasses various engineered eukaryotic microorganisms which produce methacrylic acid esters and precursors thereof as end-products. Alternatively, the engineered eukaryotic microorganisms of the invention may comprise eukaryotic microorganisms which produce methacrylic acid esters.

Microorganisms (Host Cells)

The engineered eukaryotic microorganism of the invention comprises cells which will be referred to as “hosts” or “host cells,” as they serve as hosts for the various Enzymatic Capabilities described below. A host cell can be of any species. Exemplary classes of hosts include yeasts, filamentous fungi and algae. Examples of the species which may be provided as the host include Saccharomyces cerevisiae, Pichia pastoris and Aspergillus niger.

Preferable microorganism (host cell) is yeasts. Exemplary species which may serve as hosts include microorganisms belonging to genus Saccharomyces such as Saccharomyces cerevisiae, microorganisms belonging to genus Schizosaccharomyces, microorganisms belonging to genus Pichia such as Pichia pastoris, microorganisms belonging to genus Candida such as Candida tropicalis, Candida lipolytica, Candida utilis and Candida sake, microorganisms belonging to genus Kluyveromyces, microorganisms belonging to genus Williopsis, microorganisms belonging to genus Debaryomyces, microorganisms belonging to genus Galactomyces, microorganisms belonging to genus Torulaspora, microorganisms belonging to genus Rhodotorula, microorganisms belonging to genus Yarrowia, and microorganisms belonging to genus Zygosaccharomyces. Of these, microorganisms belonging to genus Saccharomyces are preferable, and Saccharomyces cerevisiae is more preferable.

Preferred host species are those which are amenable to cell culture or which are otherwise suitable for bioproduction, wherein end-products can be synthesized in large scale production and can be inexpensively harvested and separated from the cells in which they are produced.

Transformation Methods

The host cell of the invention may be transformed to express any number of different enzymatic proteins, regulatory sequences, and other genes and gene products. Transformation may be accomplished by any means known in the art which is amenable to the selected host microorganism. In yeast, for example, alkaline cationic transformation protocols, such as lithium acetate in combination with single-stranded carrier DNA and polyethylene glycol may be used. Examples of other transformation techniques adaptable for the host cell transformation include chemical transformation methods known in the art (e.g., DEAE-dextran, polyethyleneimine, dendrimer, polybrene, calcium phosphate, lipofectin, DOTOP, Lipofectamine or CTAB/DOPE, and DOTMA); and physical transformation methods (e.g., injections, gene shock or laser-guided transduction, fine needles, and gene guns). The engineered microorganism of the invention may be transiently transformed or stably transformed.

Gene Constructs

The enzymatic gene and/or accompanying regulatory sequence introduced into the host microorganism can be configured in various ways. It will be understood that heterologous expression of a foreign gene in a host microorganism may require that codon optimizations and other sequence modifications be made in order that the gene be properly transcribed and translated in the host, as known in the art. It will also be understood that localization signals, promoters, and other elements which may be combined with the enzymatic gene sequence must be selected or altered so as to be effective in the host microorganisms, as known in the art.

Enzymatic gene introduced into the host microorganism will typically be operably linked to the downstream of a promoter sequence. In some embodiments, a constitutive promoter is utilized with the enzymatic gene to ensure constant and high level expression. Exemplary constitutive promoters include the TEF1, and GDS promoters. The genes coding for the enzyme may alternatively be placed under the control of an inducible promoter. For example, in some embodiments, one or more enzymatic capabilities of the engineered microorganism may be placed under the control of the inducible promoter known in the art and compatible with the host microorganism in order to allow for timed staging of enzymatic steps in the production of MMA end-products. For example, the GAL 10 and GAL 1 galactose inducible promoters may be used in yeast.

Localization Signals

Further, the enzymatic gene introduced into host microorganisms may also comprise a trafficking signal that directs the localization of the expression product to a specific cellular compartment. For example, a signal which localizes enzymes to the mitochondrial compartments, the cell membrane, or the chloroplast (in plant cells) may be operably linked to gene sequences coding for enzymatic proteins. The scope of the invention encompasses an engineered host wherein one or more of the enzymes introduced into the host are targeted to the mitochondria, for example, the matrix of the mitochondria. Further, the enzyme gene introduced into the host microorganism may comprise a trafficking signal which commands the localization to a specific cell compartment. For example, the matrix of the mitochondria. Examples of the signal sequence which can achieve such a targeting in a yeast include the presequence of subunit 9 of the yeast mitochondrial ATPase (Su9)(SEQ ID NO: 1) or the presequence of subunit IV of the yeast cytochrome C oxidase (Cox1) (Literature [Avalos et al., Compartmentalization of metabolic pathways in yeast mitochondria improves the production of branched-chain alcohols. Nature Biotechnol. 2013, pr; 31 (4):335-41. 2013, pr; 31(4):335-41]. Additionally, exemplary mitochondrial targeting sequences also include the presequence (residues 19-40) of the human NAOH dehydrogenase (ubiquinone) flavoprotein 2 (NOUFV2); the presequence (residues 1-20) of influenza virus protein (PB2), the presequence of yeast tryptophanyl tRNA-synthase (MSW), and the sequences described in Literature [Omura, “Mitochondria-Targeting Sequence, a Multi-Role Sorting Sequence Recognized at All Steps of Protein Import into Mitochondria,” Journal of Biochemistry. 1998, Vol. 123 Issue 6, p 1010-1016. 7p]. In some embodiments, the enzymatic gene is intended to operate in the cytosol and no trafficking moieties are utilized. In another embodiment, one or more of the expressed enzymatic proteins is designed to be secreted from the cell and gene sequences coding for such proteins are operably linked to secretion signals with direct the translated protein out of the cells.

In some embodiments, a termination sequence is utilized. For example, in yeast, CYCT transcription termination sequence may be operably linked to the enzymatic protein coding sequences to enhance proper expression in yeast cells. Other exemplary regulatory elements that can be used include the rrnB (T1) element from E. coli, the AdhT element from yeast, and the TEF1 element from yeast.

It will be understood that the enzymatic capabilities described below may be introduced by the transformation of the host microorganism with various nucleic acid constructs. Exemplary nucleic acid constructs of the invention comprise plasmids and linear nucleic acid constructs, which may comprise cloning vectors, expression cassettes, and other DNA constructs known in the art.

For a given host microorganism being transformed to express two or more enzymatic genes, such two or more enzymatic genes may be introduced into the microorganism as a single nucleic acid construct, or may be introduced in separate nucleic acid constructs. When two or more separate nucleic acid constructs are utilized, the transformation of the microorganism may occur as a single transformation event employing multiple nucleic acid constructs, or as a series of sequential transformation events to introduce the two or more genes in stages.

It will be understood that genes introduced into the host microorganism may be under the control of different promoters. For example, where the relative ratios of enzymes need to be tuned, some may be under the control of a weak promoter and some may be under the control of a stronger promoter, in order to create the desired balance of enzymatic activity in the cell. Likewise, enzymatic activity can be balanced by varying the copy number of the various enzymatic genes introduced into the host microorganism. For example a gene may be introduced as a single copy or as two, three, four or more copies, for example as end-to-end copies on a single nucleic acid construct.

Gene Equivalents

Reference to various enzymatic genes, proteins, and other genetic/protein elements is made herein. It will be understood by one of skill in the art that equivalents may be used in place of the gene and protein sequences enumerated or referenced herein. For example, variants of cited sequences may be used, including nucleic acid sequence variants and peptide sequence variants.

For example, the listed sequences may have nucleotides and/or amino acids comprising 1 or several, for example, 1 to 10, 1 to 6, 1 to 4, 1 to 3, 1 or 2 substitutions, additions, insertions or deletions, introduced thereinto.

Alternatively, genes and proteins having nucleic acid sequences or amino acid sequences having a sequence identity of 60% or more, preferably 70% or more, more preferably 80% or more, further preferably 90% or more, to the sequences mentioned are included.

Such alterations of the original sequences may be neutral or may alter (e.g. enhance) the desired enzymatic activity of the target gene. It will also be understood that gene equivalents include orthologs, paralogs, and homologs of the enumerated sequence from other species. In one embodiment, the equivalent genes of the invention comprise genes having at least 90 percent sequence identity or similarity between the wild type sequence and the variant. Further, it will be understood that functional equivalents of enumerated gene sequences may be used, such functional equivalents performing identical enzymatic functions to the enzymes or regulatory signals, while not necessarily having sequence similarity, identity or homology.

Enzymatic Capabilities

According to the present application, the engineered microorganism of the invention comprises specific “enzymatic capabilities.” Having specific enzymatic capabilities means that the microorganism has an ability to produce a specific product from a specific reactant and such an ability may be enabled by the expression of one or more enzymes which are commanded to convert the reactant to the product.

In some embodiments, one or more enzymatic capabilities possessed by the microorganism are the results of the microorganism having been transformed to express the requisite protein or multiple proteins (e.g. as in the multimeric BCKAD enzyme comprising Enzymatic Capability 2, as describe below) for such enzymatic capability. In some embodiments, all of the enzymatic capabilities possessed by an engineered microorganism of the invention are imparted by introduction of the requisite proteins by genetic transformation. In some embodiments, one or more of the enzymatic capabilities possessed by the engineered microorganism of the invention comprises a native enzymatic capability, i.e. the host microorganism's native enzymes are capable of imparting the activity without supplementation by introduced genes. For example, in some microorganisms, Enzymatic Capability 2 is possessed by the microorganism through the action of native BCKAD genes.

In one embodiment, one or more proteins introduced by the transformation are targeted to a cell compartment or organelle. In an embodiment, one or more or all proteins introduced into the microorganism by the transformation are targeted to mitochondria. In one embodiment, the mitochondrial-targeted proteins are targeted to the mitochondrial matrix. In one embodiment, the host cell is a yeast cell and the mitochondrial-targeted protein comprises a targeting moiety comprising the Su9 or Cox1 mitochondrial-targeted sequence.

The pathways for forming the MMA end-products are largely overlapping, as depicted in FIG. 1.

For example, three enzymatic steps convert valine to MAA-CoA, which can be harvested and further processed ex-vivo to create MMA. Alternatively, an MAA-CoA producing microorganism may comprise additional enzymatic capabilities that allow the MAA-CoA to be converted to 3-HIB or MMA directly. 3-HIB formed by the microorganism can be harvested and processed ex-vivo to form MMA.

While the efficient formation of MMA end-products is not found in natural microorganisms, the pathways which allow the engineered microorganism to form such products are based on valine synthetic and catabolic pathways that are common across a wide range of microorganisms. Accordingly, there is a diverse set of enzymes available to the skilled practitioner for imparting the desired enzymatic

capabilities. The specific enzymatic capabilities which allow formation of MAACoA, 3-HIB, and MMA are described hereinafter in detail. Enzymatic Capability 1: Production of 2-Oxoisovaleric Acid from Valine

A first enzymatic step in the conversion of valine to am MMA precursor is the formation of 2-oxoisovalerate from valine. This may be accomplished by the action of a branched chain aminotransferase enzyme (BCAT). In some cases, native BCAT activity or equivalent enzymatic activity is present and sufficient to produce adequate 2-oxoisovalerate from valine for the synthesis of an MMA precursor. Alternatively, host microorganisms may be transformed to express one or more BCAT or equivalent genes. Exemplary BCAT genes include the BAT1 and BAT2 genes of Saccharomyces cerevisiae. Additional BCAT genes that may be used include those listed in Table 1.

TABLE 1 BAT1 and BAT2 Genes Uniprot Gene and Protein Codes Type Gene Protein BAT1 Q6FTS6 Q6FTS6_CANGA BAT1 J8PN44 J8PN44_SACAR BAT1 C8ZA09 C8ZA09_YEAS8 BAT1 E7KPP0 E7KPP0_YEASL BAT1 E7QFU8 E7QFU8_YEASZ BAT1 E7NIT4 E7NIT4_YEASO BAT1 E7Q4X8 E7Q4X8_YEASB BAT1 B3LSW9 B3LSW9_YEAS1 BAT1 C7GN93 C7GN93_YEAS2 BAT1 BCA1 BCA1_YEAST BAT1 A6ZTB5 A6ZTB5_YEAS7 BAT1 G2WFT2 G2WFT2_YEASK BAT1 E7KDL7 E7KDL7_YEASA BAT1 C7GN93 C7GN93_YEAS2 BAT1 BCA1 BCA1_YEAST BAT1 A6ZTB5 A6ZTB5_YEAS7 BAT1 G2WFT2 G2WFT2_YEASK BAT1 L0PE74 L0PE74_PNEJ8 BAT1 B6K620 B6K620_SCHJY BAT1 BCA1 BCA1_SCHPO BAT1 Q6CAN4 Q6CAN4_YARLI BAT1 K0KC83 K0KC83_WICCF BAT1 K0KHQ6 K0KHQ6_WICCF BAT1 I2GXJ8 I2GXJ8_TETBL BAT1 I2H598 I2H598_TETBL BAT1 G8BQ96 G8BQ96_TETPH BAT1 G8BQZ4 G8BQZ4_TETPH BAT1 A7TPV1 A7TPV1_VANPO BAT1 A7TT81 A7TT81_VANPO BAT1 J7RVX9 J7RVX9_KAZNA BAT1 J7S3X0 J7S3X0_KAZNA BAT1 H2ARK1 H2ARK1_KAZAF BAT1 H2AW48 H2AW48_KAZAF BAT1 H2AYK9 H2AYK9_KAZAF BAT1 G0VL14 G0VL14_NAUCC BAT1 G0WA50 G0WA50_NAUDC BAT1 G0WHB0 G0WHB0_NAUDC BAT1 Q6FK92 Q6FK92_CANGA BAT1 Q6FTS6 Q6FTS6_CANGA BAT1 J8PN44 J8PN44_SACAR BAT1 J8Q0H7 J8Q0H7_SACAR BAT1 J8TXF1 J8TXF1_SACK1 BAT1 C8ZA09 C8ZA09_YEAS8 BAT1 C8ZBU8 C8ZBU8_YEAS8 BAT1 E7KPP0 E7KPP0_YEASL BAT1 E7KQQ1 E7KQQ1_YEASL BAT1 E7QFU8 E7QFU8_YEASZ BAT1 E7QGZ0 E7QGZ0_YEASZ BAT1 E7NIT4 E7NIT4_YEASO BAT1 E7NJL2 E7NJL2_YEASO BAT1 E7Q4X8 E7Q4X8_YEASB BAT1 E7Q5U8 E7Q5U8_YEASB BAT1 B3LQM7 B3LQM7_YEAS1 BAT1 B3LSW9 B3LSW9_YEAS1 BAT1 C7GN93 C7GN93_YEAS2 BAT1 C7GPU1 C7GPU1_YEAS2 BAT1 BCA1 BCA1_YEAST BAT1 BCA2 BCA2_YEAST BAT1 A6ZQA4 A6ZQA4_YEAS7 BAT1 A6ZTB5 A6ZTB5_YEAS7 BAT1 G2WFT2 G2WFT2_YEASK BAT1 G2WHF0 G2WHF0-YEASK BAT1 B5VKD7 B5VKD7_YEAS6 BAT1 B5VLT5 B5VLT5_YEAS6 BAT1 E7KDL7 E7KDL7_YEASA BAT1 E7KEL9 E7KEL9_YEASA BAT1 G8ZSL8 G8ZSL8_TORDC BAT1 C5E1Q1 C5E1Q1_ZYGRC BAT1 C5DFQ6 C5DFQ6_LACTC BAT1 Q6CX88 Q6CX88_KLULA BAT1 G8JWT0 G8JWT0_ERECY BAT1 Q75BE8 Q75BE8_ASHGO BAT1 C4XWX3 C4XWX3_CLAL4 BAT1 G3B9K0 G3B9K0_CANTC BAT1 A5DGQ7 A5DGQ7_PICGU BAT1 Q6BSQ6 Q6BSQ6_DEBHA BAT1 G8YUX2 G8YUX2_PICSO BAT1 A3LNY1 A3LNY1_PICST BAT1 A3LVU4 A3LVU4_PICST BAT1 G3ALC5 G3ALC5_SPAPN BAT1 G3AR71 G3AR71_SPAPN BAT1 A5DUJ5 A5DUJ5_LODEL BAT1 A5DY11 A5DY11_LODEL BAT1 H8WXC3 H8WXC3_CANO9 BAT1 H8X5S6 H8X5S6_CANO9 BAT1 G8BDW7 G8BDW7_CANPC BAT1 G8BGY0 G8BGY0_CANPC BAT1 C5M6U4 C5M6U4_CANTT BAT1 C5MJG1 C5MJG1_CANTT BAT1 B9WB98 B9WB98_CANDC BAT1 B9WEE3 B9WEE3_CANDC BAT1 Q59YS9 Q59YS9_CANAL BAT1 Q5AHX4 Q5AHX4_CANAL BAT1 Q5AHX5 Q5AHX5_CANAL BAT1 C4YIA7 C4YIA7_CANAW BAT1 C4YNT6 C4YNT6_CANAW BAT1 E7RA63 E7RA63_PICAD BAT1 F2QZT3 F2QZT3_PICP7 BAT1 C4R7A4 C4R7A4_PICPG BAT2 BAT2 L0PE74 L0PE74_PNEJ8 BAT2 B6K620 B6K620_SCHJY BAT2 BCA1 BCA1_SCHPO BAT2 Q6CAN4 Q6CAN4_YARLI BAT2 K0KC83 K0KC83_WICCF BAT2 K0KHQ6 K0KHQ6_WICCF BAT2 I2GXJ8 I2GXJ8_TETBL BAT2 I2H598 I2H598_TETBL BAT2 G8BQ96 G8BQ96_TETPH BAT2 G8BQZ4 G8BQZ4_TETPH BAT2 A7TPV1 A7TPV1_VANPO BAT2 A7TT81 A7TT81_VANPO BAT2 J7RVX9 J7RVX9_KAZNA BAT2 J7S3X0 J7S3X0_KAZNA BAT2 H2ARK1 H2ARK1_KAZAF BAT2 H2AW48 H2AW48_KAZAF BAT2 H2AYK9 H2AYK9_KAZAF BAT2 G0VL14 G0VL14_NAUCC BAT2 G0WA50 G0WA50_NAUDC BAT2 G0WHB0 G0WHB0_NAUDC BAT2 Q6FK92 Q6FK92_CANGA BAT2 Q6FTS6 Q6FTS6_CANGA BAT2 J8PN44 J8PN44_SACAR BAT2 J8Q0H7 J8Q0H7_SACAR BAT2 J8TXF1 J8TXF1_SACK1 BAT2 C8ZA09 C8ZA09_YEAS8 BAT2 C8ZBU8 C8ZBU8_YEAS8 BAT2 E7KPP0 E7KPP0_YEASL BAT2 E7KQQ1 E7KQQ1_YEASL BAT2 E7QFU8 E7QFU8_YEASZ BAT2 E7QGZ0 E7QGZ0_YEASZ BAT2 E7NIT4 E7NIT4_YEASO BAT2 E7NJL2 E7NJL2_YEASO BAT2 E7Q4X8 E7Q4X8_YEASB BAT2 E7Q5U8 E7Q5U8_YEASB BAT2 B3LQM7 B3LQM7_YEAS1 BAT2 B3LSW9 B3LSW9_YEAS1 BAT2 C7GN93 C7GN93_YEAS2 BAT2 C7GPU1 C7GPU1_YEAS2 BAT2 BCA1 BCA1_YEAST BAT2 BCA2 BCA2_YEAST BAT2 A6ZQA4 A6ZQA4_YEAS7 BAT2 A6ZTB5 A6ZTB5_YEAS7 BAT2 G2WFT2 G2WFT2_YEASK BAT2 G2WHF0 G2WHF0_YEASK BAT2 B5VKD7 B5VKD7_YEAS6 BAT2 B5VLT5 B5VLT5_YEAS6 BAT2 E7KDL7 E7KDL7_YEASA BAT2 E7KEL9 E7KEL9_YEASA BAT2 G8ZSL8 G8ZSL8_TORDC BAT2 C5E1Q1 C5E1Q1_ZYGRC BAT2 C5DFQ6 C5DFQ6_LACTC BAT2 Q6CX88 Q6CX88_KLULA BAT2 G8JWT0 G8JWT0_ERECY BAT2 Q75BE8 Q75BE8_ASHGO BAT2 C4XWX3 C4XWX3_CLAL4 BAT2 G3B9K0 G3B9K0_CANTC BAT2 A5DGQ7 A5DGQ7_PICGU BAT2 Q6BSQ6 Q6BSQ6_DEBHA BAT2 G8YUX2 G8YUX2_PICSO BAT2 A3LNY1 A3LNY1_PICST BAT2 A3LVU4 A3LVU4_PICST BAT2 G3ALC5 G3ALC5_SPAPN BAT2 G3AR71 G3AR71_SPAPN BAT2 A5DUJ5 A5DUJ5_LODEL BAT2 A5DY11 A5DY11_LODEL BAT2 H8WXC3 H8WXC3_CANO9 BAT2 H8X5S6 H8X5S6_CANO9 BAT2 G8BDW7 G8BDW7_CANPC BAT2 G8BGY0 G8BGY0_CANPC BAT2 C5M6U4 C5M6U4_CANTT BAT2 C5MJG1 C5MJG1_CANTT BAT2 B9WB98 B9WB98_CANDC BAT2 B9WEE3 B9WEE3_CANDC BAT2 Q59YS9 Q59YS9_CANAL BAT2 Q5AHX4 Q5AHX4_CANAL BAT2 Q5AHX5 Q5AHX5_CANAL BAT2 C4YIA7 C4YIA7_CANAW BAT2 C4YNT6 C4YNT6_CANAW BAT2 E7RA63 E7RA63_PICAD BAT2 F2QZT3 F2QZT3_PICP7 BAT2 C4R7A4 C4R7A4_PICPG BAT2 G1XAC5 G1XAC5_ARTOA BAT2 G3JGP7 G3JGP7_CORMM BAT2 C9STB5 C9STB5_VERA1 BAT2 G2WSU1 G2WSU1_VERDV BAT2 L2G6Q7 L2G6Q7 _COLGN BAT2 E3QRQ7 E3QRQ7_COLGM BAT2 H1VX68 H1VX68_COLHI BAT2 F7VW13 F7VW13_SORMK BAT2 Q7S699 Q7S699_NEUCR BAT2 F8MW76 F8MW76_NEUT8 BAT2 G4V0L6 G4V0L6_NEUT9 BAT2 G0SAN1 G0SAN1_CHATD BAT2 G2QRV4 G2QRV4_THITE BAT2 Q2GSR0 Q2GSR0_CHAGB BAT2 G2QEV2 G2QEV2_THIHA BAT2 F0XML2 F0XML2_GROCL BAT2 J3NH78 J3NH78_GAGT3 BAT2 G4MK83 G4MK83_MAGO7 BAT2 L8FLF5 L8FLF5_GEOD2 BAT2 K1WSP9 K1WSP9_MARBU BAT2 H0EE28 H0EE28_GLAL7 BAT2 A7ENI6 A7ENI6_SCLS1 BAT2 G2YC57 G2YC57_BOTF4 BAT2 F9X0G1 F9X0G1_MYCGM BAT2 K2SB17 K2SB17_MACPH BAT2 K2ST37 K2ST37_MACPH BAT2 Q0UN77 Q0UN77_PHANO BAT2 E4ZLK4 E4ZLK4_LEPMJ BAT2 B2VTC9 B2VTC9_PYRTR BAT2 E3RLT7 E3RLT7_PYRTT BAT2 H6BV59 H6BV59_EXODN BAT2 C1H0V7 C1H0V7_PARBA BAT2 C1GC48 C1GC48_PARBD BAT2 C0S9L5 C0S9L5_PARBP BAT2 F2TPG3 F2TPG3_AJEDA BAT2 C5JPW9 C5JPW9_AJEDS BAT2 C5G9D5 C5G9D5_AJEDR BAT2 A6RH28 A6RH28_AJECN BAT2 C0NIH3 C0NIH3_AJECG BAT2 FOU4P3 F0U4P3_AJEC8 BAT2 C6H7T8 C6H7T8_AJECH BAT2 C5FF64 C5FF64_ARTOC BAT2 E5QYK2 E5QYK2_ARTGP BAT2 F2Q3W3 F2Q3W3_TRIEC BAT2 F2S550 F2S550_TRIT1 BAT2 F2SNV1 F2SNV1_TRIRC BAT2 D4B2E4 D4B2E4_ARTBC BAT2 D4DE82 D4DE82_TRIVH BAT2 C4JT41 C4JT41_UNCRE BAT2 J3KDG0 J3KDG0_COCIM BAT2 C5P8J6 C5P8J6_COCP7 BAT2 E9D0N0 E9D0N0_COCPS BAT2 B6Q223 B6Q223_PENMQ BAT2 B6QEX9 B6QEX9_PENMQ BAT2 B8LUG0 B8LUG0_TALSN BAT2 B8MBA9 B8MBA9_TALSN BAT2 B6HRY8 B6HRY8_PENCW BAT2 K9H3Y0 K9H3Y0_PEND1 BAT2 K9FRA1 K9FRA1_PEND2 BAT2 A1CCC2 A1CCC2_ASPCL BAT2 A1CGS8 A1CGS8_ASPCL BAT2 A1CY02 A1CY02_NEOFI BAT2 B0Y5G1 B0Y5G1_ASPFC BAT2 Q4WNL4 Q4WNL4_ASPFU BAT2 C8V969 CBV969_EMENI BAT2 Q5AV02 Q5AV02_EMENI BAT2 G7X6X6 G7X6X6_ASPKW BAT2 G3Y9W8 G3Y9W8_ASPNA BAT2 A2QHM8 A2QHM8_ASPNC BAT2 Q0CHM6 Q0CHM6_ASPTN BAT2 B8NA84 B8NA84_ASPFN BAT2 I8II26 I8II26_ASPO3 BAT2 Q2UG50 Q2UG50_ASPOR BAT2 F4P9T3 F4P9T3_BATDJ BAT2 I1BVM3 I1BVM3_RHIO9 BAT2 I1C2I9 I1C2I9_RHIO9 BAT2 I1CJX9 I1CJX9_RHIO9 BAT2 F4SC44 F4SC44_MELLP BAT2 J3PTD9 J3PTD9_PUCT1 BAT2 E3KPV1 E3KPV1_PUCGT BAT2 G7DX97 G7DX97_MIXOS BAT2 A8PZH7 A8PZH7_MALGO BAT2 I2FTT8 I2FTT8_USTH4 BAT2 I2G3T8 I2G3T8_USTH4 BAT2 E6ZJK3 E6ZJK3_SPORE BAT2 E6ZRU0 E6ZRU0_SPORE BAT2 Q4P2X7 Q4P2X7_USTMA BAT2 Q4PIE8 Q4PIE8_USTMA BAT2 I4YAT5 I4YAT5_WALSC BAT2 J5SV95 J5SV95_TRIAS BAT2 K1WJA5 K1WJA5_TRIAC BAT2 E6RFZ8 E6RFZ8_CRYGW BAT2 J9VWH6 J9VWH6_CRYNH BAT2 Q55HM3 Q55HM3_CRYNB BAT2 Q5K761 Q5K761_CRYNJ BAT2 G4TBC5 G4TBC5_PIRID BAT2 G4TKH5 G4TKH5_PIRID BAT2 D8PN41 D8PN41_SCHCM BAT2 D8QEG6 D8QEG6_SCHCM BAT2 D8QKG1 D8QKG1_SCHCM BAT2 D8QKG3 D8QKG3_SCHCM BAT2 F8PKS8 F8PKS8_SERL3 BAT2 F8QC65 F8QC65_SERL3 BAT2 F8NJB4 F8NJB4_SERL9 BAT2 F8PBH0 F8PBH0_SERL9 BAT2 K5WMI9 K5WMI9_PHACS BAT2 K5XD27 K5XD27_PHACS BAT2 J4G859 J4G859_FIBRA BAT2 J4GSH9 J4GSH9_FIBRA BAT2 J4IC35 J4IC35_FIBRA BAT2 B8P088 B8P088_POSPM BAT2 B8P1C9 B8P1C9_POSPM BAT2 B8P391 B8P391_POSPM BAT2 B8P3C2 B8P3C2_POSPM BAT2 B8P3T6 B8P3T6_POSPM BAT2 B8P502 B8P502_POSPM BAT2 B8P6B4 B8P6B4_POSPM BAT2 B8P6B6 B8P6B6_POSPM BAT2 B8P6C8 B8P6C8_POSPM BAT2 B8P6D3 B8P6D3_POSPM BAT2 B8P6D7 B8P6D7_POSPM BAT2 B8P6D8 B8P6D8_POSPM BAT2 B8P6E0 B8P6E0_POSPM BAT2 B8P6E3 B8P6E3_POSPM BAT2 B8PB39 B8PB39_POSPM BAT2 B8PB40 B8PB40_POSPM BAT2 B8PB41 B8PB41_POSPM BAT2 B8PB50 B8PB50_POSPM BAT2 B8PB51 B8PB51_POSPM BAT2 B8PBJ2 B8PBJ2_POSPM BAT2 B8PBJ3 B8PBJ3_POSPM BAT2 B8PBJ9 B8PBJ9_POSPM BAT2 B8PCD3 B8PCD3_POSPM BAT2 B8PDE3 B8PDE3_POSPM BAT2 B8PDE8 B8PDE8_POSPM BAT2 B8PK93 B8PK93_POSPM BAT2 B8PK94 B8PK94_POSPM BAT2 B8PK98 B8PK98_POSPM BAT2 B8PLQ0 B8PLQ0_POSPM BAT2 B8PNG6 B8PNG6_POSPM BAT2 E2LGP7 E2LGP7_MONPE BAT2 E2LXF2 E2LXF2_MONPE BAT2 E2M281 E2M281_MONPE BAT2 A8N0B4 A8N0B4_COPC7 BAT2 A8N0V2 A8N0V2_COPC7 BAT2 B0CPH7 B0CPH7_LACBS BAT2 B0DKQ1 B0DKQ1_LACBS BAT2 K5WWN9 K5WWN9_AGABU BAT2 K5X684 K5X684_AGABU BAT2 K9HUV1 K9HUV1_AGABB BAT2 K9ICL0 K9ICL0_AGABB BAT2 D5GAR6 D5GAR6_TUBMM BAT2 D5GAR7 D5GAR7_TUBMM BAT2 C7Z586 C7Z586_NECH7 BAT2 J9MTH0 J9MTH0_FUSO4 BAT2 F9FL84 F9FL84_FUSOF BAT2 K3V6P5 K3V6P5_FUSPC BAT2 I1RYQ4 I1RYQ4_GIBZE BAT2 G9NGT1 G9NGT1_HYPAI BAT2 G0RC37 G0RC37_HYPJQ BAT2 G9N619 G9N619_HYPVG BAT2 E9DYK8 E9DYK8_METAQ BAT2 E9EQ54 E9EQ54_METAR BAT2 J5JG13 J5JG13_BEAB2

Enzymatic Capability 2: Decarboxylation of 2-Oxoisovaleric Acid to Isobutyryl-CoA

The second step in the bioproduction of MAA precursors from valine is the decarboxylation of 2-oxoisovalerate to isobutyryl-CoA. This step may be accomplished by the branched-chain α-keto acid dehydrogenase (BCKAD) complex. The BCKAD complex comprises four subunits, the E1 subunit, the E2 subunit, the E3 subunit, and a dihydrolipoamide dehydrogenase.

In some cases, sufficient BCKAD or equivalent activity is present in the host microorganism to efficiently convert 2-oxoisovalerate to isobutyryl-CoA. For example, as described in Example 1, engineered yeast cells produced ample MMA precursors without the introduction of BCKAD genes, demonstrating that sufficient native activity may be present in the host.

Alternatively, the host may be transformed to express one or more enzymes that perform the decarboxylation of 2-oxoisovalerate to isobutyryl-CoA. For example, the host microorganism may be engineered to express the four constituents of the BCKAD complex. For example, the four genes encoding the Pseudomonas aeruginosa-BCKAD complex may be introduced into the host to impart or augment native BCKAD activity. These genes include: the bkdA1 gene, encoding E1 (Genbank Accession No. NP250937), bkdA2 gene, encoding E3 (Genbank Accession No. NP_250938), bkdB gene, encoding E2 (Genbank Accession No. NP250939) and IpdV gene, encoding dihydrolipoamide dehydrogenase (Genbank Accession No. NP250940). Alternatively, the host may be transformed to express a bkdA1 gene selected from Table 2, a bkdA2 gene selected from Table 3, a bkdB gene selected from Table 4, and an IpdV gene selected from Table 5.

TABLE 2 bkdA1 Genes Genome Gene ID# Locus Tag Stigmatella aurantiaca DW4/3-1 310822118 STAUR_4871 Glaciecola nitratireducens FR1064 348029317 GNIT_901 Corallococcus coralloides DSM 2259 383456734 COCOR_04759 Sinorhizobium fredii HH103 378827583 SFHH103_02998 Alteromonas macleodi ATCC 27126 406596861 MASE_09530 Pseudomonas fluorescens F113 378950264 PSF113_2346 Sinorhizobium meliloti SM11 384537524 SM11_chr3104 Brucella suis VBI22 376278492 BSVBI22_B0518 Shewanella baltica BA175 386324712 Sbal175_2270 Brucella melitensis M5-90 384213034 BM590_B0496 Alteromonas macleodi str. ‘English 407683852 AMEC673_09775 Channel 673’ Marinobacter hydrocarbonoclasticus 387814424 MARHY2809 ATCC 49840 Pseudomonas aeruginosa NCGM2.S1 386066185 NCGM2_3256 Pseudomonas fluorescens A506 387894499 PflA506_3337 Alteromonas macleodi str. ‘Balearic 407687775 AMBAS45_09980 Sea AD45’ Sinorhizobium meliloti BL225C 384530814 SinmeB_2782 Thermus thermophilus JL-18 386359631 TtJL18_0174 Shewanella baltica OS117 386341194 Sbal117_2345 Brucella melitensis NI 384446660 BMNI_II0489 Brucella canis HSK A52141 376276775 BCA52141_II0445 Pseudomonas aeruginosa M18 386058856 PAM18_2793 Pseudomonas pulida S16 339488698 PPS_3805 Shewanella baltica OS678 378708518 Sbal678_2203 Alteromonas macleodi str. ‘Black Sea 407700100 AMBLS11_09275 11’ Marinobacter adhaerens HP15 385329802 HP15_61 Burkholderia cepacia GG4 402556979 GEM_2217 Brucella melitensis M28 384410135 BM28_B0497 Sphingobium sp. SYK-6 347527350 SLG_09650 Pseudomonas pulida DOT-T1E 397697834 T1E_5099 Burkholderia pseudomallei 1026b 386856107 BP1026B_II2449 Oceanimonas sp. GK1 374335112 GU3_06465 Shewanella putrefaciens 200 386313862 Sput200_2106 Brucella pinnipedialis B2/94 340792252 BP1_II505 384223054 BS1330_II0519 Pelaglbacterium halotolerans B2 357384137 KKY_1077 Sinorhizobium fredii USDA 257 3983S5221 USDA257_c54050 Shewanella woodyi ATCC 51908 170726629 Swoo_2278 Shewanella denitrificans OS217 91793142 Sden_1786 Shewanella oneidensis MR-1 24373885 SO_2339 Shewanella halifaxensis HAW-EB4 167624158 Shal_2234 Anaeromyxobacter dehalogenans 2CP-C 86158863 Adeh_2441 Parachlamydia acanthamoebae UV7 338174253 PUV_02590 Agrobacterium tumefaciens str. C58 159185754 Atu3473 Pseudoalteromonas atlantica T6c 109898353 Patl_2036 Pseudomonas fluorescens SBW25 229591396 PFLU3964 Sorangium cellulosum ‘So ce 56’ 162449842 sce1571 Pseudomonas fluorescens Pf-5 70729902 PFL_2534 Oceanithermus profundus DSM 14977 313680637 Ocepr_1750 Bradyrhizobium japonicum USDA 110 27381442 blr6331 Brucella melitensis ATCC 23457 225686316 BMEA_B0497 Deinococcus deserti VCD115 226357394 Deide_23440 Pseudomonas putida KT2440 26991090 PP_4401 Shewanelle loihica PV-4 127512853 Shew_1925 Shewanelle baltica OS185 153000673 Shew185_2149 Thermus thermophilus HB8 55980198 TTHA0229 Brucella suis 1330 23500271 BRA0524 Burkholderia pseudomallei 1106a 126456753 BURPS1106A_A3067 Mesorhizobium ciceri biovar biserrulae 319780620 Mesci_0881 WSM1271 Polaromonas sp. JS666 91786184 Bpro_0273 Burkholderia glumae BGR1 238023732 bglu_2g02640 Alicycliphilus denitrificans BC 319761347 Alide_0628 Brucella abortus A13334 376271257 BAA13334_II01347 Alicycliphilus denitrificans K601 330823222 Alide2_0593 Burkholderia pseudomallei 668 126444383 BURPS668_A3193 Burkholderia gladioli BSR3 330819430 bgla_2g03040 Sphingomonas wittichii RW1 148555060 Swit_2145 Pseudoalteromonas sp. SM9913 315126681 PSM_A1605 Brucella suis ATCC 23445 163844682 BSUIS_B0519 Myxococcus xanthus DK 1622 108760073 MXAN_4564 Shewanella sediminis HAW-EB3 157375466 Ssed_2329 Pseudomonas putida F1 148546693 Pput_1453 Brucella abortus bv. 1 str. 9-941 62317615 BruAb2_0700 Burkholderia mallei SAVP1 121597380 BMASAVP1_1036 Pseudomonas aeruginosa DK2 392984204 PADK2_14040 Brevundimonas subvibrioides ATCC 302384438 Bresu_3332 15264 Sphingobium chlorophenolicum L-1 334344849 Sphch_1205 Shewanella sp. MR-7 114047443 Shewmr7_1947 Hahella chejuensis KCTC 2396 83646416 HCH_03687 Shewanella violacea DSS12 294140813 SVI_2042 Novosphingobium aromaticivorans 87199993 Saro_1976 DSM 12444 Pseudomonas putida ND6 395447960 YSA_07994 Thermus thermophilus HB27 46200059 TTC1757 Burkholderia ambifaria MC40-6 172060189 BamMC406_1134 Burkholderia pseudomallei 1710b 76819484 BURPS1710b_A1411 Shewanella frigidimarina NCIMB 400 114563106 Sfri_1935 Hirschia baltica ATCC 49814 254293976 Hbal_1614 Thermoplasma acidophilum DSM 1728 16082407 Ta1438 Mesorhizobium opportunistum 337265440 Mesop_0911 WSM2075 Sinorhizobium meliloti 1021 15966685 SMc03201 Burkholderia mallei NCTC 10247 126447708 BMA10247_A2302 Bdellovibrio bacteriovorus HD100 42522535 Bd0972 Shewanella baltica OS155 126174438 Sbal_2222 Bordetella petrii DSM 12804 163857824 Bpet3511 Brucella canis ATCC 23365 161620586 BCAN_B0522 Brucella ovis ATCC 25840 148557918 BOV_A0455 Colwellia psychrerythraea 234H 71281526 CPS_1582 Mesorhizobium loti MAFF303099 13473769 mll4473 Pseudomonas putida BIRD-1 386011042 PPUBIRD1_1441 Stigmatella aurantiaca DW4/3-1 310822118 STAUR_4871 Pseudomonas aeruginosa PA7 152985298 PSPA7_2994 Anaeromyxobacter sp. K 197121831 AnaeK_1422 Maricaulls maris MCS10 114569255 Mmar10_0704 Anaeromyxobacter dehalogenans 2CP-1 220916623 A2cp1_1517 Burkholderia cenocepacia J2315 206559589 BCAL1212 Pseudomonas brassicacearum subsp. 330810242 PSEBR_a3378 brassicacearum NFM421 Brucella melitensis bv. 1 str. 16M 17989093 BMEII0748 Shewanella baltica OS195 160875312 Sbal195_2199 Burkholderia sp. 383 78065831 Bcep18194_A4360 Haliangium ochraceum DSM 14365 262196423 Hoch_3237 Variovorax paradoxus S110 239814017 Vapar_1010 Achromobacter xylosoxidans A8 311105811 AXYL_02629 Ruegeria sp. TM1040 99082618 TM1040_2778 Pseudoalteromonas haloplanktis 77360573 PSHAa1632 TAC125 Pseudomonas fluorescens Pf0-1 77459685 Pfl01_3463 Sphingobium japonicum UT26S 294011285 SJA_C1-12990 Shewanella sp. W3-18-1 120598931 Sputw3181_2123 Waddlia chondrophila WSU 86-1044 297620705 wcw_0464 Shewanella baltica OS223 217973406 Sbal223_2235 Rubrobacter xylanophilus DSM 9941 108805282 Rxyl_2480 Idiomarina loihiensis L2TR 56460781 IL1680 Caulobacter sp. K31 167645834 Caul_1870 Shewanelle pealeana ATCC 700345 157962067 Spea_2246 Agrobacterium sp. H13-3 332716274 AGROH133_11609 Shewanella amazonensis SB2B 119774845 Sama_1709 Brucella abortus S19 189022867 BAbS19_II06670 Novosphingobium sp. PP1Y 334142274 PP1Y_AT28841 Burkholderia mallei NCTC 10229 124381521 BMA10229_1322 Burkholderia thailandensis E264 83717236 BTH_II2304 Halomonas elongata DSM 2581 307544960 HELO_2370 Pseudomonas aeruginosa LESB58 218891779 PLES_30571 Sinorhizobium fredii NGR234 227823512 NGR_c29890 Alteromonas macleodii str. ‘Deep 332141378 MADE_1009900 ecotype’ Brucella microti CCM 4915 256015303 BMI_II518 Sphingopyxis alaskensis RB2256 103486817 Sala_1331 Shewanella sp. ANA-3 117920575 Shewana3_2131 Shewanella putrefaciens CN-32 146292983 Sputcn32_1885 Burkholderia pseudomallei K96243 53723291 BPSS2273 Burkholderia mallei ATCC 23344 53716061 BMAA2013 Burkholderia cenocepacia AU 1054 107022318 Bcen_0762 Pseudomonas aeruginosa UCBPP- 116050194 PA14_35530 PA14 Thermoplasma volcanium GSS1 13540933 TVN0102 Pseudomonas entomophila L48 104782857 PSEEN3853 Burkholderia cenocepacia MC0-3 170732566 Bcenmc03_1216 Burkholderia cenocepacia HI2424 116689265 Bcen2424_1243 Alteromonas sp. SN2 333893048 ambt_07965 Shewanella sp. MR-4 113970365 Shewmr4_2028 Brucella melitensis biovar Abortus 83269598 BAB2_0715 2308 Ramlibacter tataouinensis TTB310 337278681 Rta_10480 Ochrobactrum anthropi ATCC 49188 153010872 Oant_3551 Erythrobacter litoralis HTCC2594 85373858 ELI_05155 Pseudomonas putida GB-1 167034957 PputGB1_3962 Burkholderia ambifaria AMMD 115351176 Bamb_1123 Sinorhizobium medicae WSM419 150398024 Smed_2826 Anaeromyxobacter sp. Fw109-5 153004855 Anae109_1993 Pusillimonas sp.T7-7 332284089 PT7_0836 Shewanella piezotolerans WP3 212635405 swp_2606 Rhodoferax ferrireducens T118 89902318 Rfer_3554 Glaciecola sp. 4H-3-7 + YE-5 332306594 Glaag_2232 Marinobacter aquaeolei VT8 120554306 Maqu_1382 Ferrimonas balearica DSM 9799 308050068 Fbal_2358 Sinorhizobium meliloti AK83 334317690 Sinme_2988 Pseudomonas putida W619 170722905 PputW619_3742

TABLE 3 bkdA2 genes Genome Gene ID# Locus Tag Arthrobacter sp. FB24 116669943 Arth_1382 Stigmatella aurantiaca DW4/3-1 310822117 STAUR_4870 Chlamydia trachomatis G/9301 385242630 CTG9301_01750 Glaciecola nitratireducens 348029316 GNIT_1900 FR1064 Chlamydia psittaci NJ1 406593263 B712_0357 Listeria monocytogenes 405758053 LMOSLCC2479_1066 SLCC2479 Pseudonocardia dioxanivorans 331700311 Psed_6609 CB1190 Belliella baltica DSM 15883 390941876 Belba_0214 Staphylococcus aureus subsp. 384870059 SAT0131_01610 aureus T0131 Mycoplasma bovis HB0801 392429694 Mbov_0103 Listeria ivanovii subsp. ivanovii 347548454 LIV_0998 PAM 55 Muricauda ruestrlngensls DSM 344204449 Murru_3149 13258 Lactococcus garvieae ATCC 347520637 LCGT_0031 49156 Salinibacter ruber M8 294506482 SRM_00667 Chlamydia trachomatis F/SW5 389859784 FSW5_3451 Listeria monocytogenes M7 386026370 LMM7_1083 Bacillus subtilis subsp. subtilis str. RO-NN-1 384176023 I33_2482 Listeria monocytogenes 404413140 LMOSLCC7179_1034 SLCC7179 Sinorhizobium fredii HH103 378827584 SFHH103_02999 Alteromonas macleodii ATCC 406596862 MASE_09535 27126 Staphylococcus aureus subsp. 379021299 M013TW_1532 aureus M013 Bacteroides fragilis 638R 375357937 BF638R_1637 Mycobacterium chubuense NBB4 392417338 Mycch_3528 Enterococcus hirae ATCC 9790 392989735 EHR_12860 Streptomyces bingchenggensis 374988200 SBI_05444 BCW-1 Pseudomonas fluorescens F113 378950263 PSF113_2345 Staphylococcus aureus subsp. 385781802 MS7_1534 aureus 11819-97 Staphylococcus aureus subsp. 387780609 SARLGA251_14230 aureus LGA251 Staphylococcus aureus subsp. 379014725 SAVC_06830 aureus VC40 Streptomyces sp. SirexAA-E 345000792 SACTE_3240 Staphylococcus aureus subsp. 384547750 SAOV_1517 aureus FD133 Mycoplasma bovis Hubel-1 339320624 MMB_0097 Sinorhizobium meliloti SM11 384537525 SM11_chr3105 Granulicella mallensis 374310265 AciX8_1322 MP5ACTX8 Lactococcus garvieae Lg2 385832000 LCGL_0031 Chlamydia trachomatis Sweden2 386262693 SW2_3451 Paenibacillus polymyxa M1 386041287 PPM_2597 Bacillus amyloliquefaciens LL3 384164972 LL3_02590 Chlamydia psittaci CP3 406592170 B711_0381 Listeria monocytogenes J0161 386046715 LMOG_00667 Lactobacillus casei BD-II 385823210 LCBD_1514 Listeria monocytogenes 404410298 LMOSLCC5850_1059 SLCC5850 Melissococcus plutonius DAT561 379727537 MPD5_0997 Pyrobaculum oguniense TE7 379004029 Pogu_1067 Chlamydia trachomatis E/SW3 389858908 ESW3_3451 Nocardiopsis alba ATCC BAA- 403509181 B005_1702 2165 Chlamydia trachomatis F/SW4 389858032 FSW4_3451 Listeria monocytogenes 405755132 LMOSLCC2540_1053 SLCC2540 Staphylococcus aureus subsp. 386729217 ST398NM01_1582 aureus 71193 Brucella suis VBI22 376278493 BSVBI22_B0519 Synechocystis sp. PCC 6803 383490833 SYNPCCN_0870 substr. PCC-N Mycobacterium massiliense str. 397680860 MYCMA_2661 GO 06 Listeria monocytogenes L99 386007777 lmo4a_1062 Exiguobacterium antarcticum B7 407477698 Eab7_1859 Listeria monocytogenes 404407509 LMOSLCC2376_1026 SLCC2376 Ignavibacterium album JCM 385810230 IALB_1650 16511 Staphylococcus lugdunensis 385784340 SLUG_13960 N920143 Bacillus amyloliquefaciens subsp. 384266014 BANAU_2384 plantarum YAU B9601-Y2 Chlamydia trachomatis G/11074 385246240 G11074_01750 Corynebacterium variabile DSM 340793900 CVAR_0937 44702 Niastella koreensis GR20-10 375149638 Niako_6452 Bacteriovorax marinus SJ 374289801 BMS_3178 Mycoplasma pneumoniae FH 385326972 MPNE_0454 Lactobacillus rhamnosus GG 385827987 LRHM_1267 NC_017482 Shewanelle baltica BA175 386324711 Sbal175_2269 Acidithiobacillus caldus SM-1 340782068 Atc_1326 Streptomyces cattleya NRRL 386356697 SCATT_30500 8057 = DSM 46488 NC_017586 Brucella melitensis M5-90 384213035 BM590_B0497 Chlamydia trachomatis D-EC 385243547 CTDEC_0340 Staphylococcus 386319228 SPSE_1284 pseudintermedius ED99 Lactobacillus casei LC2W 385820009 LC2W_1479 Bacillus amyloliquefaciens subsp. 375362935 BACAU_2245 plantarum CAU B946 Alteromonas macleodii str. 407683853 AMEC673_09780 ‘English Channel 673’ Staphylococcus aureus subsp. 384864739 ECTR2_1368 aureus ECT-R 2 Mycoplasma genitalium M2288 402552594 CM5_01605 Aequorivita sublithincola DSM 390955426 Aeqsu_2718 14238 Streptomyces hygroscopicus 386841339 SHJG_5256 subsp. jingnangensis 5008 Actinoplanes missouriensis 431 383775247 AMIS_770 Chlamydia trachomatis A2497 376282346 CTR_3381 NC_016798 Thermus sp. CCB_US3_UF1 384439515 TCCBUS3UF1_11210 Listeria monocytogenes serotype 404286467 LMOSLCC2482_1100 7 str. SLCC2482 Staphylococcus aureus subsp. 384867510 HMPREF0772_11624 aureus TCH60 Lactobacillus rhamnosus ATCC 385835175 LRHK_1311 8530 Marinobacter 387814425 MARHY2010 hydrocarbonoclasticus ATCC 49840 Pseudomonas aeruginosa 386066186 NCGM2_3257 NCGM2.S1 Bacillus amyloliquefaciens TA208 384160048 BAMTA208_12325 Pseudomonas fluorescens A506 387894500 PflA506_3338 Alteromonas macleodii str. 407687776 AMBAS45_09985 ‘Balearic Sea AD45’ Sinorhizobium meliloti BL225C 384530815 SinmeB_2763 Mycoplasma hyopneumoniae 168 385334342 MHP168_185 Thermus thermophilus JL-18 386359630 T1JL18_0173 Listeria monocytogenes 405752275 LMOSLCC2378_1071 SLCC2378 Listeria monocytogenes Finland 386053324 LMLG_2335 1998 Leuconostoc sp. C2 339490188 LGMK_00030 Shewanella baltica OS117 386341193 Sbal117_2344 Brucella melitensis NI 384446661 BMNI_II0490 Lactobacillus buchneri CD034 406026601 LBUCD034_0782 Brucella canis HSK A52141 376276774 BCA52141_II0442 Bacillus amyloliquefaciens XH7 384169111 BAXH7_02513 Pseudomonas aeruginosa M18 386058855 PAM18_2792 Enterococcus faecium Aus0004 383328412 EFAU004_01093 Tetragenococcus halophilus 352517702 TEH_15280 NBRC 12172 Terriglobus roseus DSM 18391 390958199 Terro_2360 Terriglobus roseus DSM 18391 390957858 Terro_1994 Deinococcus gobiensis I-0 386855294 DGo_CA0086 Mycoplasma genitalium M6320 402552088 CM1_01650 Staphylococcus aureus 04-02981 387150660 SA2981_1475 Saprospira grandis str. Lewin 379730055 SGRA_1936 Chlamydia trachomatis A2497 385270024 CTO_0369 NC_017437 Staphylococcus aureus subsp. 386831127 SAEMRSA15_14370 aureus HO 5096 0412 Paenibacillus mucilaginosus 3016 379723636 PM3016_5961 Pseudomonas putida S16 339488699 PPS_3806 Shewanella baltica OS678 378708519 Sbal678_2204 Chlamydia trachomatis E/150 385245317 E150_01785 Mycoplasma genitallum M6282 402551583 CM3_01735 Alteromonas macleodii str. ‘Black 407700101 AMBLS11_09280 Sea 11’ Arthrobacter sp. Rue61a 403526503 ARUE_c14360 Chlamydia trachomatis G/9768 385239854 G9768_01750 Lactobacillus salivarius CECT 385839942 HN6_00131 5713 Thermus thermophilus 384430364 Ththe16_0161 SG0.5JP17-16 Chlamydla trachomatis G/11222 385240777 G11222_01755 Marinobacter adhaerens HP15 385329804 HP15_63 Burkholderia cepacia GG4 402566978 GEM_2216 Synechocystis sp. PCC 6803 383324949 SYNPCCP_0870 substr. PCC-P Streptomyces flavogriseus ATCC 357412428 Sfla_3226 33331 Actinoplanes sp. SE50/110 386845148 ACPL_194 Synechocystis sp. PCC 6803 383321780 SYNGTI_0871 substr. GT-I Brucella melitensis M28 384410136 BM28_B0498 Listeria monocytogenes 10403S 386043379 LMRG_00515 Sphingobium sp. SYK-6 347527351 SLG_09660 Enterococcus faecium DO 389868409 HMPREF0351_11226 Paenibacillus mucilaginosus K02 386726385 B2K_30300 Enterococcus faecalis 62 384518455 EF62_1805 Listeria monocytogenes ATCC 405749410 LMOATCC19117_1075 19117 Listeria monocytogenes FSL R2- 386049981 LMKG_01869 561 Streptomyces violaceusniger Tu 345008026 Strvi_0312 4113 Pseudomonas putida DOT-T1E 397697835 T1E_5100 Burkholderia pseudomallei 1026b 386866106 BP1026B_II2447 Staphylococcus aureus subsp. 384862119 SAA6008_01486 aureus str. JKD6008 Listeria monocytogenes 386731808 MUO_05540 07PF0776 Paenibacillus terrae HPL-003 374323888 HPL003_20280 Mycoplasma pneumoniae 309 377822707 MPNA3920 Staphylococcus aureus subsp. 387602857 SAPIG1582 aureus S0385 Staphylococcus aureus subsp. 387143124 SATW20_15130 aureus TW20 Oceanimonas sp. GK1 374335113 GU3_06470 Shewanella putrefaciens 200 386313861 Sput200_2105 Brucella pinnipedialis B2/94 340792253 BPI_II506 384223055 BS1330_II0520 Chlamydia trachomatls D-LC 385244427 CTDLC_0340 Kitasatospora setae KM-6054 357390777 KSE_38660 Pelagibacterium halotolerans B2 357384138 KKY_1078 Sinorhizobium fredii USDA257 398355222 USDA257_c54060 Enterococcus faecalis D32 397699743 EFD32_1166 Bacillus amyloliquefaciens Y2 387899033 MUS_2694 Chlamydia trachomatis L2c 339626009 CTL2C_951 Leuconostoc mesenteroides 381336327 MI1_03380 subsp. mesenteroides J18 Chlamydia trachomatis E/11023 385241710 E11023_01775 Melioribacter roseus P3M 397691247 MROS_2258 Mycoplasma genitalium M2321 402551095 CM9_01625 Akkermansia muciniphila ATCC 187735388 Amuc_0886 BAA-835 Mycoplasma crocodyli MP145 294155659 MCRO_0411 Acidobacterium sp. MP5ACTX9 322435297 Aclx9_1679 Shewanella woodyi ATCC 51908 170726629 Swoo_2279 Shewanella denitrificans OS217 91793143 Sden_1787 Nitratifractor salsuginis DSM 319956742 Nitsa_0997 16511 Prochlorococcus marinus subsp. 33240216 Pro0766 marinus str. CCMP1375 Shewanella oneidensis MR-1 24373887 SO_2340 Lactobacillus sakei subsp. sakei 81428694 LSA1084 23K Aster yellows witches'-broom 85057417 AYWB_137 phytoplasma AYWB Shewanella halifaxensis HAW- 167624157 Shal_2233 EB4 Anaeromyxobacter dehalogenans 86158250 Adeh_1826 2CP-C Parachlamydia acanthamoebae 338174252 PUV_02580 UV7 Rhodococcus erythropolis PR4 226307480 RER_39930 Paenibacillus sp. JDR-2 251796660 Pjdr2_2651 Listeria seeligeri serovar 1/2b str. 289434314 lse_0947 SLCC3954 Agrobacterium tumefaciens str. 15891465 Atu_3472 C58 Pseudoalteromonas atlantica T6c 109898354 Patl_2037 Chlamydia muridarum Nigg 15835233 TC0618 Pseudomonas fluorescens 229591397 PFLU3965 SBW25 Sorangium cellulosum ‘So ce 56’ 162449841 sce1570 Melissococcus plutonius ATCC 332686440 MPTP_0946 35311 Pseudomonas fluorescens Pf-5 70729901 PFL_2533 Bacillus subtilis subsp. spizizenii 305675052 BSUW23_11885 str. W23 Oceanithermus profundus DSM 313680638 Ocepr_1751 14977 Bradyrhizoblum Japonicum USDA 27381443 blr6332 110 Lactobacillus salivarius UCC118 90961136 LSL_0154 Paenibacillus mucilaginosus 337750577 KNP414_06348 KNP414 Croceibacter atlanticus 298208751 CA2559_10943 HTCC2559 Brucella melitensis ATCC 23457 225686317 BMEA_B0498 Gramella forsetii KT0803 120435970 GFO_1616 Arthrobacter arilaitensis Re117 308176762 AARI_09800 Candidatus Koribacter versatilis 94968813 Acid345_1786 Ellin345 Deinococcus deserti VCD115 226357395 Deide_23450 Chloroflexus sp. Y-400-fl 222525095 Chy400_1833 Stackebrandtia nassauensis DSM 291303508 Snas_6069 44728 Pseudomonas putida KT2440 26991091 PP_4402 Synechococcus sp. JA-2-3B′a(2- 86609223 CYB_1765 13) Shewanella loihica PV-4 127512854 Shew_1926 Synechococcus sp. JA-3-3Ab 86606954 CYA_2326 Shewanella baltica OS185 153000674 Shew185_2150 Enterococcus faecalis V583 29375921 EF1354 Staphylococcus epidermidis 57866997 SERP1077 RP62A Staphylococcus aureus subsp. 148268001 SaurJH9_1575 aureus JH9 Mycoplasma pneumoniae M129 13508131 MPN392 Staphylococcus aureus subsp. 57650473 SACOL1561 aureus COL Staphylococcus epidermidis 27468115 SE1197 ATCC 12228 Thermus thermophilus HB8 55980199 TTHA0230 Brucella suis 1330 23500272 BRA0525 Aeropyrum pernix K1 14601550 APE_1674 Microbacterium testaceum 323360061 MTES_3613 StLB037 Onion yellows phytoplasma OY-M 39939087 PAM_601 Marinomonas sp. MWYL1 152997133 Mmwyl1_3123 Renibacterium salmoninarum 163839305 RSal33209_0547 ATCC 33209 Beutenbergia cavernae DSM 229822443 Bcav_3966 12333 Burkholderia pseudomallei 1106a 126455889 BURPS1106A_A3066 Mesorhizobium ciceri biovar 319780621 Mesci_0882 biserrulae WSM1271 Arthrobacter phenanthrenivorans 325962775 Asphe3_13730 Sphe3 Streptomyces scabiei 87.22 290958943 SCAB_45171 Exiguobacterium sp. AT1b 229916237 EAT1b_0506 Deinococcus proteolyticus MRP 325284178 Deipr_1971 Polaromonas sp. JS666 91786185 Bpro_0274 Streptomyces cattleya NRRL 357400644 SCAT_3060 8057 = DSM 46488 NC_016111 Mycoplasma conjunctivae 240047197 MCJ_000680 HRC/581 Burkholderia glumae BGR1 238023733 bglu_2g02650 Alicycliphilus denitrificans BC 319761348 Alide_0629 Staphylococcus aureus subsp. 161509745 USA300HOU_1518 aureus USA300_TCH1516 Brucella abortus A13334 376271256 BAA13334_II01344 Alicycliphilus denitrificans K601 330823223 Alide2_0594 Chlamydia trachomatis 434/Bu 166154552 CTL0594 Burkholderia pseudomallei 668 126444265 BURPS668_A3192 Burkholderia gladioli BSR3 330819431 bgla_2g03050 Staphylococcus aureus subsp. 49486353 SAS1455 aureus MSSA476 Sphingomonas wittichii RW1 148555059 Swit_2144 Staphylococcus lugdunensis 289550713 SLGD_01399 HKU09-01 Pseudoalteromonas sp. SM9913 315126680 PSM_A1604 Brucella suis ATCC 23445 163844683 BSUIS_B0520 Truepera radiovictrix DSM 17093 297622991 Trad_0747 Mycoplasma pulmonis UAB CTIP 15829234 MYPU_7630 Micromonospora aurantiaca 302864656 Micau_0148 ATCC 27029 Prochlorococcus marinus str. MIT 124023323 P9303_16211 9303 Shewanella sediminis HAW-EB3 157375465 Ssed_2328 Listeria innocua Clip11262 16800114 lin1045 Chitinophaga pinensis DSM 2588 256419854 Cpin_0808 Mycoplasma hyopneumoniae 72080456 MHP7448_0116 7448 Pseudomonas putida F1 148546692 Pput_1452 Brucella abortus bv. 1 str. 9-941 62317614 BruAb2_0699 Burkholderia mallei SAVP1 121597989 BMASAVP1_1035 Pseudomonas aeruginosa DK2 392984203 PADK2_14035 Frankia alni ACN14a 111219575 FRAAL0070 Brevundimonas subvibrioides 302384436 Bresu_3330 ATCC 15264 Sphingobium chlorophenolicum L-1 334344848 Sphch_1204 Shewanella sp. MR-7 114047444 Shewmr7_1948 Hahella chejuensis KCTC 2396 83646415 HCH_03686 Shewanella violacea DSS12 294140814 SVI_2043 Staphylococcus camosus subsp. 224476625 Sca_1138 carnosus TM300 Bacteroides fragilis NCTC 9343 60681130 BF1636 Novosphingobium 87199992 Saro_1975 aromaticivorans DSM 12444 Deinococcus geothermalis DSM 94986436 Dgeo_2339 11300 Cyanothece sp. ATCC 51142 172039560 cce_4647 Pseudomonas putida ND6 395447959 YSA_07992 Leuconostoc gasicomitatum LMG 300173681 LEGAS_1380 18811 Intrasporangium calvum DSM 317123421 Intca_0249 43043 Thermus thermophilus HB27 46200058 TTC1756 Burkholderia ambifaria MC40-6 172060190 BamMC406_1135 Chloroflexus aggregans DSM 219848986 Cagg_2096 9485 Burkholderia pseudomallei 1710b 76818778 BURPS1710b_A1410 Mycoplasma agalactiae 291320032 MAGa1010 404489824 BLi02581 Shewanella frigidimarina NCIMB 114563107 Sfri_1936 400 Staphylococcus aureus subsp. 379795877 SAMSHR1132_13570 aureus MSHR1132 Staphylococcus aureus subsp. 269203146 SAAV_1509 aureus ED98 Hirschia baltica ATCC 49814 254293977 Hbal_1615 Thermoplasma acidophilum DSM 16082406 Ta1437 1728 Mesorhizobium opportunistum 337265441 Mesop_0912 WSM2075 Sinorhizobium meliloti 1021 15966686 SMc03202 Salinispora tropica CNB-440 145592674 Strop_0108 Burkholderia mallei NCTC 10247 126446124 BMA10247_A2301 Bdellovibrio bacteriovorus HD100 42522536 Bd0974 Shewanella baltica OS155 126174437 Sbal_2221 Candidatus Phytoplasma mali 194246556 ATP_00155 Frankia symbiont of Datisca 336180266 FsymDg_4471 glomerata Candidatus Phytoplasma 197294721 PAa_0687 australiense Staphylococcus 319892499 SPSINT_1210 pseudintermedius HKU10-03 Bordetella petrii DSM 12804 163857823 Bpet3510 Deinococcus maricopensis DSM 320333704 Deima_1097 21211 Listeria monocytogenes L312 406703829 LMOL312_1054 Verrucosispora maris AB-18-032 330464990 VAB18032_05035 Brucella canis ATCC 23365 161620587 BCAN_B0523 Brucella ovis ATCC 25840 148558303 BOV_A0456 Colwellia psychrerythraea 34H 71277818 CPS_1583 Mesorhizobium loti MAFF303099 13473768 mll4472 Cellulomonas fimi ATCC 484 332671066 Celf_2562 Pseudomonas putida BIRD-1 386011041 PPUBIRD1_1440 Maribacter sp. HTCC2170 305666956 FB2170_11881 Pyrobaculum calidifontis JCM 126460012 Pcal_1404 11548 Stigmatella aurantiaca DW4/3-1 310822117 STAUR_4870 Staphylococcus aureus subsp. 49483766 SAR1594 aureus MRSA252 Mycoplasma agalactiae PG2 148377362 MAG_0940 Pseudomonas aeruginosa PA7 152988941 PSPA7_2993 Lactobacillus buchneri NRRL B- 331701105 Lbuc_0737 30929 Photorhabdus asymbiotica 253990152 PAU_02673 Staphylococcus aureus subsp. 156979835 SAHV_1504 aureus Mu3 Staphylococcus aureus subsp. 384550344 SAA6159_01452 aureus JKD6159 Bacillus amyloliquefaciens DSM 7 308174192 BAMF_2301 Anaeromyxobacter sp. K 197122440 AnaeK_2034 Maricaulis maris MCS10 114569256 Mmar10_0705 Anaeromyxobacter dehalogenans 220917207 A2cp1_2104 2CP-1 Burkholderia cenocepacia J2315 206559590 BCAL1213 Pseudomonas brassicacearum 330810243 PSEBR_a3379 subsp. brassicacearum NFM421 Staphylococcus aureus subsp. 15924506 SAV1516 aureus Mu50 Lactobacillus rhamnosus Lc 705 258539526 LC705_01335 Rhodococcus opacus B4 226360693 ROP_12790 Leuconostoc kimchil IMSNU 296110720 LKI_02945 11154 Brucella melitensis bv. 1 str. 16M 17989092 BMEII0747 Shewanella baltica OS195 160875313 Sbal195_2200 Staphylococcus aureus subsp. 88195323 SAOUHSC_01612 aureus NCTC 8325 16330037 sll1721 Cellulophaga algicola DSM 319955314 Cetal_3836 14237 Leuconostoc citreum KM20 170016783 LCK_00425 Burkholderia sp. 383 78065832 Bcep18194_A4361 Marivirga tractuosa DSM 4126 313676905 Ftrac_2815 Haliangium ochraceum DSM 262196422 Hoch_3236 14365 Synechocystis sp. PCC 6803 384436100 SYNGTS_0871 Variovorax paradoxus S110 239814018 Vapar_1011 Achromobacter xylosoxidans A8 311105812 AXYL_02630 Ruegeria sp. TM1040 99082617 TM1040_2777 Arthrobacter chlorophenolicus A6 220912170 Achl_1400 Chloroflexus aurantiacus J-10-fl 163847254 Caur_1692 Haliscomenobacter hydrossis 332665004 Halhy_3056 DSM 1100 Pseudoalteromonas haloplanktis 77360572 PSHAa1631 TAC125 Thermoproteus uzoniensis 768- 327310924 TUZN_1028 20 Pseudomonas fluorescens Pf0-1 77459686 Pfl01_3464 Lactobacillus casei ATCC 334 116494795 LSEI_1306 Lactobacillus casei str. Zhang 301066361 LCAZH_1300 Sphingobium japonicum UT26S 294011286 SJA_C1-13000 Cyanothece sp. PCC 8801 218247666 PCC8801_2883 Shewanella sp. W3-18-1 120598930 Sputw3181_2122 Propionibacterium freudenreichii 297625421 PFREUD_02200 subsp. shermanll CIRM-BIA1 Bacillus subtilis subsp. subtilis str. 16079460 BSU24040 168 Nocardiopsis dassonvillei subsp. 297564330 Ndas_5418 dassonvillei DSM 43111 Waddlia chondrophila WSU 86- 297620706 wcw_0465 1044 Shewanella baltica OS223 217973405 Sbal223_2234 Frankia sp. Ccl3 86738780 Francci3_0057 Bacillus licheniformis ATCC 52080942 BL01505 14580 Idiomarina loihiensis L2TR 56460780 IL1679 Cellulomonas flavigena DSM 296131225 Cfla_3399 20109 Bacillus clausii KSM-K16 56964215 ABC2450 Caulobacter sp. K31 167645835 Caul_1871 Enterococcus faecalis OG1RF 384513106 OG1RF_11142 Thermobaculum terrenum ATCC 269925215 Tter_0094 BAA-798 Roseiflexus castenholzii DSM 156743005 Rcas_3062 13941 Prochlorococcus marinus str. MIT 33862891 PMT0618 9313 Shewanella pealeana ATCC 157962066 Spea_2245 700345 Agrobacterium sp. H13-3 332716273 AGROH133_11608 Rhodococcus equi 103S 312139113 REQ_16940 Shewanella amazonensis SB2B 119774846 Sama_1710 Bacillus cellulosilyticus DSM 317129371 Bcell_2667 2522 Brucella abortus S19 189022866 BAbS19_II06660 Novosphingobium sp. PP1Y 334142273 PP1Y_AT28832 Listeria monocytogenes HCC23 217964854 LMHCC_1574 Leuconostoc mesenteroides 116617850 LEUM_0738 subsp. mesenteroides ATCC 8293 Thermobifida fusca YX 72160585 Tfu_0181 Deinococcus radiodurans R1 15805071 DR_0030 Burkholderia mallei NCTC 10229 124382700 BMA10229_1321 Burkholderia thailandensis E264 83716737 BTH_II2303 Pedobacter heparinus DSM 2366 255533705 Phep_3824 Halomonas elongata DSM 2581 307544961 HELO_2371 Pseudomonas aeruginosa 218891778 PLES_30561 LESB58 Sinorhizobium fredii NGR234 227823513 NGR_c29900 Alteromonas macleodii str. ‘Deep 332141379 MADE_1009905 ecotype’ Brucella microti CCM 4915 256015304 BMI_II519 Meiothermus ruber DSM 1279 291296694 Mrub_2321 Sphingopyxis alaskensis RB2256 103486816 Sala_1330 Chlamydia trachomatis 237804687 CTB_3381 B/TZ1A828/OT Chlamydia trachomatis D/UW- 15605063 CT340 3/CX Listeria monocytogenes serotype 46907285 LMOf2365_1074 4b str. F2365 Mycoplasma genitalium G37 12045129 MG_273 Shewanella sp. ANA-3 117920574 Shewana3_2130 Shewanella putrefaciens CN-32 146292984 Sputcn32_1886 Burkholderia pseudomallei 53723290 BPSS2272 K96243 Synechococcus sp. PCC 7002 170077278 SYNPCC7002_A0655 Mycobacterium ulcerans Agy99 118619019 MUL_3774 Arthrobacter aurescens TC1 119960874 AAur_1522 Burkholderia mallei ATCC 23344 53716062 BMAA2012 Saccharomonospora viridis DSM 257057824 Svir_38870 43017 Mycobacterium sp. MCS 108800591 Mmcs_3625 Staphylococcus aureus subsp. 150394068 SaurJH1_1608 aureus JH1 Burkholderia cenocepacia AU 107022319 Bcen_0763 1054 Meiothermus silvanus DSM 9946 297565575 Mesil_1135 Chlamydia trachomatis L2b/UCH- 166155427 CTLon_0592 1/proctitis Parabacteroides distasonis ATCC 150007337 BDI_0687 8503 Staphylococcus haemolyticus 70726400 SH1399 JCSC1435 Lactobacillus fermentum IFO 184155615 LAF_1139 3956 Bacillus amyloliquefaciens FZB42 154686663 RBAM_022320 Staphylococcus aureus subsp. 21283198 MW1469 aureus MW2 Frankia sp. EAN1pec 158318985 Franean1_7268 Pseudomonas aeruginosa 116050195 PA14_35520 UCBPP-PA14 Thermoplasma volcanium GSS1 13540932 TVN0101 Paenibacillus polymyxa E681 308069294 PPE_02531 Lactobacillus casei BL23 191638310 LCABL_15370 Pseudomonas entomophila L48 104782858 PSEEN3854 Terriglobus saanensis SP1PR4 320107218 AciPR4_2010 Staphylococcus aureus subsp. 87160136 SAUSA300_1465 aureus USA300_FPR3757 Lactobacillus rhamnosus GG 258508316 LGG_01321 NC_013198 Staphylococcus aureus RF122 82751121 SAB1389c Burkholderia cenocepacia MC0-3 170732567 Bcenmc03_1217 Thermus scotoductus SA-01 320449332 TSC_c02390 Paracoccus denitrificans PD1222 119384344 Pden_1604 Thermomonospora curvata DSM 269124599 Tcur_0329 43183 Burkholderia cenocepacia HI2424 116689266 Bcen2424_1244 Oenococcus oeni PSU-1 116490425 OEOE_0329 Roseiflexus sp. RS-1 148656538 RoseRS_2416 Listeria monocytogenes 08-5578 284801385 LM5578_1136 Alteromonas sp. SN2 333893049 ambt_07970 Shewanella sp. MR-4 113970364 Shewmr4_2027 Streptomyces avermitilis MA- 29830920 SAV_4377 4680 Staphylococcus aureus subsp. 15927097 SA1347 aureus N315 Bacteroides fragilis YCH46 53712912 BF1622 Thermobispora bispora DSM 296271282 Tbis_3331 43833 Paenibacillus sp. Y412MC10 261406246 GYMC10_2402 Erysipelothrix rhusiopathiae str. 336066645 ERH_1409 Fujisawa Pyrobaculum arsenaticum DSM 145591407 Pars_1188 13514 Mycoplasma bovis PG45 313678231 MBOVPG45_0105 Brucella melitensis biovar Abortus 83269597 BAB2_0714 2308 Rhodococcus jostii RHA1 111018576 RHA1_ro01577 Ramlibacter tataouinensis 337278682 Rta_10490 TTB310 Ochrobactrum anthropi ATCC 153010873 Oant_3552 49188 Erythrobacter litoralis HTCC2594 85373859 ELI_05160 Bacillus subtilis BSn5 321311885 BSn5_02555 Pseudomonas putida GB-1 167034958 PputGB1_3963 Actinosynncma mirum DSM 256379011 Amir_5002 43827 Cyanothece sp. PCC 7822 307152588 Cyan7822_2730 Mycobacterium sp. KMS 119869730 Mkms_3698 Burkholderia ambifaria AMMD 115351177 Bamb_1124 Sinorhizobium medicae WSM419 150398025 Smed_2827 Mycobacterium vanbaalenii PYR-1 120405039 Mvan_4085 Chloroherpeton thalassium ATCC 193214148 Ctha_0429 35110 Lysinibacillus sphaericus C3-41 169826944 Bsph_1364 Salinibacter ruber DSM 13855 83816020 SRU_0576 Bacteroides thetaiotaomicron VPI- 29345722 BT_0312 5482 Chlamydia trachomatis A/HAR-13 76789066 CTA_0369 Anaeromyxobacter sp. Fw109-5 153004856 Anae109_1994 Chlamydia trachomatis 237802765 JALI_3381 B/Jali20/OT Simkania negevensis Z 338732442 SNE_A05470 Acidobacterium capsulatum 225874704 ACP_3156 ATCC 51196 Micromonospora sp. L5 315500949 ML5_0131 Listeria monocytogenes EGD-e 16803093 lmo1053 Cyanothece sp. PCC 8802 257060998 Cyan8802_3213 Prochlorococcus marinus str. MIT 159903256 P9211_07151 9211 Carnobacterium sp. 17-4 328957242 CAR_c09190 Saccharopolyspora erythraea 134100488 SACE_3953 NRRL 2338 Pusillimonas sp. T7-7 332284090 PT7_0837 Synechococcus elongatus PCC 56751371 syc1362_d 6301 Shewanella piezotolerans WP3 212635404 swp_2605 Streptosporangium roseum DSM 271970151 Sros_8973 43021 Paenibacillus polymyxa SC2 310642336 PPSC2_c2888 ‘Nostoc azollae’ 0708 298492752 Aazo_4540 Rhodoferax ferrireducens T118 89902317 Rfer_3553 Streptomyces griseus subsp. 182437559 SGR_3766 griseus NBRC 13350 Pyrobaculum aerophilum str. IM2 18313490 PAE2646 Mycobacterium abscessus ATCC 169631990 MAB_4917c 19977 Salinispora arenicola CNS-205 159035781 Sare_0108 Listeria monocytogenes 08-5923 284994527 LM5923_1090 Sphaerobacter thermophilus 269929376 Sthe_3476 DSM 20745 Mycoplasma hyopneumoniae 232 54020422 mhp264 Marinithermus hydrothermalis 328950234 Marky_0709 DSM 14884 Herpetosiphon aurantiacus ATCC 159899111 Haur_2592 23779 Listeria weishimeri serovar 6b str. 116872447 lwe1029 SLCC5334 Glaciecola sp. 4H-3-7 + YE-5 332306593 Glaag_2231 Cellulophaga lytica DSM 7489 325287820 Celly_2922 Mycoplasma hyopneumoniae J 71893469 MHJ_0112 Marinobacter aquaeolei VT8 120554305 Maqu_1381 Mycobacterium sp. JLS 126436207 Mjls_3630 Picrophilus torridus DSM 9790 48477620 PTO0548 Staphylococcus aureus subsp. 151221634 NWMN_1422 aureus str. Newman Ferrimonas balearica DSM 9799 308050067 Fbal_2357 Listeria monocytogenes serotype 226113671 Lm4b_01073 4b str. CLIP 80459 Sinorhizobium meliloti AK83 334317691 Sinme_2989 Pseudomonas putida W619 170722906 PputW619_3743

TABLE 4 bkdB genes Genome Gene ID# Locus Tag Stigmatella aurantiaca 310821917 STAUR_4868 DW4/3-1 Mycoplasma putrefaciens 344205274 MPUT_0557 KS1 Spirochaeta thermophila 386347079 Spith_1347 DSM 6578 Glaciecola nitratireducens 348029315 GNIT_1899 FR1064 Chlamydia psittaci NJ1 406593425 B712_0521 Listeria monocytogenes 405758054 LMOSLCC2479_1067 SLCC2479 Bacillus coagulans 36D1 347750662 Bcoa_0221 Desulfosporosinus orientis 374996775 Desor_4331 DSM 765 Streptococcus suis D9 386584873 SSUD9_1864 Lactococcus lactis subsp. 389853269 LLNZ_00355 cremoris NZ9000 Staphylococcus aureus 384869628 SAT0131_01132 subsp. aureus T0131 Sulfobacillus acidophilus 339629489 TPY_3237 TPY Listeria ivanovii subsp. 347548455 LIV_0999 ivanovii PAM 55 Corallococcus coralloides 383455711 COCOR_03727 DSM 2259 Streptococcus suis ST1 389857346 SSUST1_1724 Mycoplasma hyorhinis 385858736 SRH_03265 MCLD Listeria monocytogenes M7 386026371 LMM7_1084 Zymomonas mobilis subsp. 384411955 Zmob 1035 mobilis ATCC 10988 Bacillus subtilis subsp. 384175196 I33_1640 subtilis str. RO-NN-1 Listeria monocytogenes 404413141 LMOSLCC7179_1035 SLCC7179 Sinorhizobium fredii HH103 378827585 SFHH103_03000 Alteromonas macleodli 406596863 MASE_09540 ATCC 27126 Sulfobacillus acidophilus 379007393 Sulac_1676 DSM 10332 Staphylococcus aureus 379020802 M013TW_1027 subsp. aureus M013 Mycoplasma gallisepticum 385325594 MGAH_0162 str. R (high) Bacteroides fragilis 638R 375357936 BF638R_1636 Chlamydophila psittaci 384454502 CPS0B_0520 02DC15 Haloferax mediterranei 389848366 HFX_2954 ATCC 33500 Chlamydophila psittaci 384451574 CPS0A_0523 01DC11 Mycobacterium canettii CIPT 340627509 MCAN_25341 140010059 Mycoplasma hyorhinis GDL-1 378835985 MYM_0544 Mycoplasma leachii 392388985 MLEA_004800 99/014/6 Streptomyces 374988188 SBI_05432 bingchenggensis BCW-1 Mycobacterium tuberculosis 392387133 UDA_2495c UT205 Pseudomonas fluorescens 378950262 PSF113_2344 F113 Staphylococcus aureus 385781323 MS7_1052 subsp. aureus 11819-97 Staphylococcus aureus 387780208 SARLGA251_10080 subsp. aureus LGA251 Staphylococcus aureus 379014288 SAVC_04635 subsp. aureus VC40 Streptomyces sp. SirexAA-E 345000808 SACTE_3256 Staphylococcus aureus 384547281 SAOV_1039 subsp. aureus ED133 Cardinium endosymbiont 406024948 CAHE_0047 cPer1 of Encarsia pergandiella Flavobacterium 347536377 FBFL15_1500 branchiophilum FL-15 Methanocella conradii 383320309 Mtc_1892 HZ254 Sinorhizobium meliloti SM11 384537526 SM11_chr3106 Pyrobaculum sp. 1860 374327309 P186_1853 Acetobacter pasteurianus 384042231 APA12_12160 IFO 3283-12 Paenibacillus polymyxa M1 386041286 PPM_2596 Bacillus amyloliquefaciens 384163956 LL3_01566 LL3 Chlamydia psittaci CP3 406592339 B711_0552 Mycobacterium tuberculosis 385999275 MTCTRI2_2541 CTRI-2 Listeria monocytogenes 386046716 LMOG_00666 J0161 Bacillus megaterium WSH- 384048055 BMWSH_3882 002 Lactobacillus casei BD-II 385823211 LCBD_1515 Acetobacter pasteurianus 384059731 APA26_12160 IFO 3283-26 Listeria monocytogenes 404410299 LMOSLCC5850_1060 SLCC5850 Pyrobaculum oguniense TE7 379004030 Pogu_1068 Amycolatopsis mediterranei 384152886 RAM_38890 S699 NC_017186 Thermococcus sp. 4557 341582347 GQS_06320 Streptococcus macedonicus 374337769 SMA_0798 ACA-DC 198 Nocardiopsis alba ATCC 403509526 B005_2061 BAA-2165 Bacillus cereus NC7401 375285965 BCN_3871 Listeria monocytogenes 405755133 LMOSLCC2540_1054 SLCC2540 Staphylococcus aureus 386728775 ST398NM01_1092 subsp. aureus 71193 Brucella suis VBI22 376278494 BSVBI22_B0520 Mycobacterium tuberculosis 383308271 MRGA327_15390 RGTB327 Mycobacterium massiliense 397678695 MYCMA_0457 str. GO 06 Listeria monocytogenes L99 386000778 lmo4a_1063 Exiguobacterium 407477697 Eab7_1858 antarcticum B7 Rhodothermus marinus 345303310 Rhom172_1451 SG0.5JP17-172 Listeria monocytogenes 404407510 LMOSLCC2376_1027 SLCC2376 Leptospira interrogans 386074087 LIF_A1623 serovar Lai str. IPAV Acetobacter pasteurianus 384057090 APA22_12160 IFO 3283-22 Mycobacterium tuberculosis 386005399 MRGA423_15610 RGTB423 Staphylococcus lugdunensis 385784712 SLUG_17750 N920143 Bacillus amyloliquefaciens 384265011 BANAU_1381 subsp. plantarum YAU B9601-Y2 Mycobacterium tuberculosis 397674399 RVBD_2495c H37Rv Mycoplasma pneumoniae 385326971 MPNE_0453 FH Mycobacterium bovis BCG 378772231 BCGMEX_2507c str. Mexico Lactobacillus rhamnosus 385827988 LRHM_1268 GG NC_017482 Shewanella baltica BA175 386324710 Sbal175_2268 Streptococcus infantarius 379705200 Sinf_0850 subsp. infantarius CJ18 Streptomyces cattleya NRRL 386356696 SCATT_30490 8057 = DSM 46488 NC_017586 Streptococcus suis GZ1 386578648 SSGZ1_1656 Geobacillus 375008018 GTCCBUS3UF5_12370 thermoleovorans CCB_US3_UF5 Brucella melitensis M5-90 384213036 BM590_B0498 Staphylococcus 386319627 SPSE_1696 pseudintermedius ED99 Lactobacillus casei LC2W 385820010 LC2W_1480 Bacillus amyloliquefaciens 375362108 BACAU_1418 subsp. plantarum CAU B946 Alteromonas macleodii str. 407683854 AMEC673_09785 ‘English Channel 673’ Chlamydophila psittaci 384453523 CPS0C_0525 C19/98 Staphylococcus aureus 384864322 ECTR2_950 subsp. aureus ECT-R 2 Streptococcus agalactiae 406709369 A964_0883 GD201008-001 Streptococcus thermophilus 386086666 STND_0986 ND03 Streptococcus gallolyticus 386337523 SGGB_0854 subsp. gallolyticus ATCC 43143 Streptomyces hygroscopicus 386841328 SHJG_5245 subsp. jinggangensis 5008 Thermus sp. 384440433 TCCBUS3UF1_20450 CCB_US3_UF1 Listeria monocytogenes 404286468 LMOSLCC2482_1101 serotype 7 str. SLCC2482 Streptococcus suis S735 403062262 YYK_07845 Staphylococcus aureus 384868023 HMPREF0772_12137 subsp. aureus TCH60 Lactobacillus rhamnosus 385835176 LRHK_1312 ATCC 8530 Pseudomonas aeruginosa 386066187 NCGM2_3258 NCGM2.S1 Bacillus anthracis str. H9401 386737860 H9401_3987 Bacillus amyloliquefaciens 384159556 BAMTA208_09845 TA208 Pseudomonas fluorescens 387894501 PflA506_3339 A506 Chlamydophila psittaci 6BC 384450574 G5O_0512 Alteromonas macleodii str. 407687777 AMBAS45_09990 ‘Balearic Sea AD45’ Sinorhizobium meliloti 384530816 SinmeB_2764 BL225C Bacillus sp. JS 386758176 MY9_1599 Mycoplasma 385334664 MHP168_513 hyopneumoniae 168 Thermus thermophilus JL-18 386359627 TtJL18_0170 Listeria monocytogenes 405752276 LMOSLCC2378_1072 SLCC2378 Listeria monocytogenes 386053325 LMLG_2963 Finland 1998 Acetobacter pasteurianus 384050748 APA03_12160 IFO 3283-03 Leuconostoc sp. C2 339490189 LGMK_00035 Shewanella baltica OS117 386341192 Sbal117_2343 Brucella melitensis NI 384446662 BMNI_II0491 Oscillibacter valericigenes 350270533 OBV_21370 Sjm18-20 Lactococcus lactis subsp. 385837009 llh_0200 cremoris A76 Mycoplasma gallisepticum 385326188 MGF_2592 str. F Mycobacterium africanum 339632521 MAF_25100 GM041182 Alicyclobacillus 384134351 TC41_0586 acidocaldarius subsp. acidocaldarius Tc-4-1 Lactobacillus buchneri 406026602 LBUCD034_0783 CD034 Brucella canis HSK A52141 376276773 BCA52141_II0441 Bacillus amyloliquefaciens 384168610 BAXH7_02008 XH7 Pseudomonas aeruginosa 386058854 PAM18_2791 M18 halophilic archaeon DL31 345006003 Halar_2851 Deinococcus gobiensis I-0 386855296 DGo_CA0088 Acetobacter pasteurianus 384053856 APA07_12160 IFO 3283-07 Mycoplasma genitalium 402552087 CM1_01645 M6320 Staphylococcus aureus 04- 387150237 SA2981_1052 02981 Mycoplasma gallisepticum 401769395 HFMG01WIA_3531 WI01_2001.043-13-2P Mycoplasma gallisepticum 401766360 HFMG94VAA_3682 VA94_7994-1-7P Staphylococcus aureus 386830631 SAEMRSA15_09250 subsp. aureus HO 5096 0412 Paenibacillus mucilaginosus 379723635 PM3016_5960 3016 Pseudomonas putida S16 339488700 PPS_3807 Amycolatopsis mediterranei 399541273 AMES_7457 S699 V2 NC_018266 Shewanella baltica OS678 378708520 Sbal678_2205 Streptococcus suis A7 386588918 SSUA7_1659 Micavibrio aeruginosavorus 347758064 MICA 1302 ARL 13 Alteromonas macleodii str. 407700102 AMBLS11_09285 ‘Black Sea 11’ Arthrobacter sp. Rue61a 403526504 ARUE_c14370 Lactobacillus salivarius 385839943 HN6_00132 CECT 5713 Thermus thermophilus 384430361 Ththe16_0158 SG0.5JP17-16 Marinobacter adhaerens 385329805 HP15_64 HP15 Mycoplasma gallisepticum 401767116 HFMG95NCA_3609 NC95_13295-2-2P Burkholderia cepacia GG4 402566977 GEM_2215 Mycobacterium tuberculosis 392431895 TBXG_001463 KZN 605 Streptomyces flavogriseus 357412429 Sfla_3227 ATCC 33331 Brucella melitensis M28 384410137 BM28_B0499 Listeria monocytogenes 386043380 LMRG_00516 10403S Sphingobium sp. SYK-6 347527352 SLG_09670 Zymomonas mobilis subsp. 397676636 ZZ6_0757 mobilis ATCC 29191 Mycoplasma gallisepticum 401770149 HFMG06NCA_3594 NC06_2006.080-5-2P Bacillus thuringiensis 384181787 YBT020_19515 serovar finitimus YBT-020 Bacillus cereus F837/76 376267867 bcf_19725 Paenibacillus mucilaginosus 386726384 B2K_30295 K02 Mycobacterium tuberculosis 385995424 CCDC5079_2299 CCDC5079 Listeria monocytogenes 405749411 LMOATCC19117_1076 ATCC 19117 Listeria monocytogenes FSL 386049982 LMKG_01868 R2-561 Natrinema sp. J7-2 397771820 NJ7G_0034 Streptomyces violaceusniger 345008014 Strvi_0300 Tu 4113 Pseudomonas putida DOT- 397697836 T1E_5101 T1E Burkholderia pseudomallei 386866105 BP1026B_II2446 1026b Staphylococcus aureus 384861690 SAA6008_01050 subsp. aureus str. JKD6008 Listeria monocytogenes 386731809 MUO_05545 07PF0776 Solibacillus silvestris 393201879 SSIL_3152 StLB046 Paenibacillus terrae HPL- 374323889 HPL003_20285 003 Mycoplasma pneumoniae 377822706 MPNA3910 309 Streptococcus suis D12 386586927 SSUD12_1813 Streptococcus suis JS14 386580721 SSUJS14_1797 Staphylococcus aureus 387602368 SAPIG1092 subsp. aureus S0385 Staphylococcus aureus 387142707 SATW20_10900 subsp. aureus TW20 Oceanimonas sp. GK1 374335114 GU3_06475 Shewanella putrefaciens 386313860 Sput200_2104 200 Brucella pinnipedialis B2/94 340792254 BPI_II507 384223056 BS1330_II0521 Streptococcus thermophilus 386344721 STH8232_1236 JIM 8232 Kitasatospora setae KM- 357390884 KSE_39730 6054 Pelagibacterium 357384139 KKY_1079 halotolerans B2 Mycobacterium tuberculosis 385991805 CCDC5180_2271 CCDC5180 Sinorhizobium fredii USDA 398355223 USDA257_c54070 257 Enterococcus faecalis D32 397699744 EFD32_1167 Acetobacter pasteurianus 384119099 APA42C_12160 IFO 3283-01-42C Bacillus amyloliquefaciens 387897996 MUS_1555 Y2 Mycobacterium tuberculosis 375295688 TBSG_01487 KZN 4207 Leuconostoc mesenteroides 381336328 MI1_03385 subsp. mesenteroides J18 Solitalea canadensis DSM 387791039 Solca_1867 3403 Mycoplasma gallisepticum 401768648 HFMG01NYA_3671 NY01_2001.047-5-1P Mycoplasma genitalium 402551094 CM9_01620 M2321 Caldicellulosiruptor 146295908 Csac_0872 saccharolyticus DSM 8903 Mycoplasma crocodyli 294155660 MCRO_0412 MP145 Shewanella woodyi ATCC 170726630 Swoo_2280 51908 Streptomyces coelicolor 21222239 SCO3829 A3(2) Shewanella denitrificans 91793144 Sden_1788 OS217 Prochlorococcus marinus 33239853 Pro0401 subsp. marinus str. CCMP1375 Streptococcus suis BM407 253756264 SSUBM407_1708 Shewanella oneidensis MR-1 24373888 SO_2341 Bacillus anthracis str. CDC 227816744 BAMEG_4223 684 Lactobacillus sakei subsp. 81428693 LSA1083 sakei 23K Aster yellows witches'- 85057418 AYWB_138 broom phytoplasma AYWB Shewanella halifaxensis 167624156 Shal_2232 HAW-EB4 Anaeromyxobacter 86158249 Adeh_1825 dehalogenans 2CP-C Parachlamydia 338174251 PUV_02570 acanthamoebae UV7 Rhodococcus erythropolis 226309469 RER_59840 PR4 Bacillus halodurans C-125 15615216 BH2653 Paenibacillus sp. JDR-2 251796661 Pjdr2_2652 Listeria seeligeri serovar 289434315 Ise_0948 1/2b str. SLCC3954 Agrobacterium tumefaciens 159185753 Atu3471 str. C58 Pseudoalteromonas 109898355 Patl_2038 atlantica T6c Chlamydia muridarum Nigg 15835136 TC0518 Pseudomonas fluorescens 229591398 PFLU3966 SBW25 Sorangium cellulosum ‘Soce 162449840 sce1569 56’ Halopiger xanaduensis SH-6 336252525 Halxa_1119 Melissococcus plutonius 332686441 MPTP_0947 ATCC 35311 Synechococcus sp. CC9605 78213526 Syncc9605_2009 Streptococcus suis 146321685 SSU98_1838 98HAH33 Pseudomonas fluorescens 70729900 PFL_2532 Pf-5 Bacillus subtilis subsp. 305674187 BSUW23_07510 spizizenii str. W23 Rhodospirillum centenum 209963468 RC1_0121 SW Bradyrhizobium japonicum 27381444 blr6333 USDA 110 Lactobacillus salivarius 90961137 LSL_0155 UCC118 Wolbachia endosymbiont of 190571193 WPa_0791 Culex quinquefasciatus Pel Paenibacillus mucilaginosus 337750576 KNP414_06347 KNP414 Haloferax volcanii DS2 292657068 HVO_2960 Brucella melitensis ATCC 225686318 BMEA_B0499 23457 Lactobacillus reuteri DSM 148543865 Lreu_0633 20016 Bacillus pumilus SAFR-032 157692138 BPUM_1357 Bacillus cereus G9842 218899126 BCG9842_B1167 Streptococcus agalactiae 25010951 gbs0897 NEM316 Chloroflexus sp. Y-400-fl 222524728 Chy400_1455 Pseudomonas putida 26991092 PP_4403 KT2440 Synechococcus sp. JA-2- 86608594 CYB_1116 3B′a(2-13) Haloterrigena turkmenica 284166851 Htur_3595 DSM 5511 Shewanella lolhica PV-4 127512855 Shew_1927 Mycobacterium tuberculosis 148662331 MRA_2521 H37Ra Synechococcus sp. JA-3- 86605452 CYA_0742 3Ab Shewanella baltica OS185 153000675 Shew185_2151 Enterococcus faecalis V583 29375922 EF1355 Staphylococcus epidermidis 57866608 SERP0682 RP62A Staphylococcus aureus 148267588 SaurJH9_1155 subsp. aureus JH9 Mycoplasma pneumoniae 13508130 MPN391 M129 Staphylococcus aureus 57651704 SACOL1104 subsp. aureus COL Staphylococcus epidermidis 27467711 SE0793 ATCC 12228 Thermus thermophilus HB8 55980201 TTHA0232 Brucella suis 1330 23500273 BRA0526 Aeropyrum pernix K1 14601549 APE_1671 Microbacterium testaceum 323358111 MTES_1663 StLB037 Gemmatimonas aurantiaca T 226227399 GAU_1993 27 Bacillus cereus AH820 218905101 BCAH820_3985 Onion yellows phytoplasma 39939088 PAM_602 OY-M Thermaerobacter 317122504 Tmar_1671 marianensis DSM 12885 332287420 CPSIT_0516 Mycoplasma leachii PG50 313665161 MSB_A0275 Burkholderia pseudomallei 126456596 BURPS1106A_A3065 1106a Leifsonia xyli subsp. xyli str. 50955930 Lxx25050 CTCB07 Treponema azotonutricium 333995417 TREAZ_3439 ZAS-9 Geobacillus sp. Y412MC61 261419258 GYMC61_1834 Mesorhizobium ciceri biovar 319780622 Mesci_0883 biserrulae WSM1271 Arthrobacter 325964389 Asphe3_30510 phenanthrenivorans Sphe3 Streptomyces scabiei 87.22 290958955 SCAB_45291 Exiguobacterium sp. AT1b 229918500 EAT1b_2787 Wolbachia endosymbiont 58585004 Wbm0747 strain TRS of Brugia malayi Lactobacillus plantarum 308180958 LPST_C1776 subsp. plantarum ST-III Ilyobacter polytropus DSM 310779675 Ilyop_1889 2926 Bacillus anthracis str. Ames 30264042 BA_4182 Polaromonas sp. JS666 91786186 Bpro_0275 Streptomyces cattleya NRRL 357400643 SCAT_3059 8057 = DSM 46488 NC_016111 Halobacterium sp. NRC-1 15791042 VNG2219G Chlamydophila psittaci RD1 392376657 Cpsi_4681 Mycoplasma conjunctivae 240047198 MCJ_000690 HRC/581 Bacillus cereus E33L 52141519 BCZK3729 Coprothermobacter 206896349 COPRO5265_0853 proteolyticus DSM 5265 Mycobacterium tuberculosis 253798425 TBMG_01476 KZN 1435 Geobacillus 138894594 GTNG_0924 thermodenitrificans NG80-2 Burkholderia glumae BGR1 238023734 bglu_2g02660 Alicycliphilus denitrificans 319761349 Alide_0630 BC Staphylococcus aureus 161509278 USA300HOU_1038 subsp. aureus USA300_TCH1516 Bacillus thuringiensis str. Al 118479182 BALH_3593 Hakam Bacillus thuringiensis 384188034 CT43_CH3976 serovar chinensis CT-43 Brucella abortus A13334 376271255 BAA13334_II01343 Bacillus thuringiensis 49481607 BT9727_3713 serovar konkuklan str. 97-27 Fluviicola taffensis DSM 327403295 Fluta_1300 16823 Alicycliphilus denitrificans 330823224 Alide2_0595 K601 Geobacillus 336236213 Geoth_2861 thermoglucosidasius C56- YS93 Alicyclobacillus 258510469 Aaci_0455 acidocaldarius subsp. acidocaldarius DSM 446 Prochlorococcus marinus 78778785 PMT9312_0400 str. MIT 9312 Streptococcus suis 146319493 SSU05_1839 05ZYH33 Anaerolinea thermophila 320160832 ANT_14280 UNI-1 Bartonella tribocorum CIP 163868060 Btr_0863 105476 Burkholderia pseudomallei 126445162 BURPS668_A3191 668 Burkholderia gladioli BSR3 330819432 bgla_2g03060 Zymomonas mobilis subsp. 56551406 ZMO0510 mobilis ZM4 Staphylococcus aureus 49485933 SAS1030 subsp. aureus MSSA476 Sphingomonas wittichii RW1 148553703 Swit_0780 Staphylococcus lugdunensis 289551093 SLGD_01780 HKU09-01 Chlamydophila pecorum 330444490 G5S_0830 E58 Staphylococcus 73663003 SSP1694 saprophyticus subsp. saprophyticus ATCC 15305 Natrialba magadii ATCC 289581338 Nmag_1666 43099 Pseudoalteromonas sp. 315126679 PSM_A1603 SM9913 Nautilia profundicola AmH 224372801 NAMH_0770 Brucella suis ATCC 23445 163844684 BSUIS_B0521 Prochlorococcus marinus 126695762 P9301_04241 str. MIT 9301 Mycoplasma pulmonis UAB 15829233 MYPU_7620 CTIP Micromonospora aurantiaca 302869527 Micau_5080 ATCC 27029 Prochlorococcus marinus 124023822 P9303_21291 str. MIT 9303 Shewanella sediminis HAW- 157375464 Ssed_2327 EB3 Listeria innocua Clip11262 16800115 lin1046 Mycoplasma synovlae 53 71894296 MS53_0274 Bacillus cereus ATCC 14579 30022059 BC3971 Mycoplasma 72080837 MHP7448_0506 hyopneumoniae 7448 Pseudomonas putida F1 148546691 Pput_1451 Brucella abortus bv. 1 str. 9- 62317613 BruAb2_0698 941 Burkholderia mallei SAVP1 121596563 BMASAVP1_1034 Mesoplasma florum L1 50364856 Mfl041 Pseudomonas aeruginosa 392084202 PADK2 14030 DK2 Mycoplasma penetrans HF-2 26553962 MYPE5100 Frankia alni ACN14a 111222643 FRAAL3226 Brevundimonas 302384435 Bresu_3329 subvibrioides ATCC 15264 Thermococcus sibiricus MM 242399616 TSIB_1641 739 Myxococcus fulvus HW-1 338535470 LILAB_29225 Modestobacter marinus 389866831 MODMU_5238 Sphingobium 334342793 Sphch_3287 chlorophenolicum L-1 Shewanella sp. MR-7 114047445 Shewmr7_1949 Hahella chejuensis KCTC 83646414 HCH_03685 2396 Shewanella violacea DSS12 294140815 SVI_2044 Mycobacterium bovis 31793675 Mb2523c AF2122/97 Synechococcus sp. CC9902 78184239 Syncc9902_0662 Staphylococcus carnosus 224476210 Sca_0721 subsp. carnosus TM300 Bacteroides fragilis NCTC 60681129 BF1635 9343 Lactobacillus fermentum 385812453 LC40_0741 CECT 5716 Novosphingobium 87199991 Saro_1974 aromaticivorans DSM 12444 Geobacillus sp. WCH70 239826459 GWCH70_0954 Geobacter sp. M18 322421393 GM18_3918 Deinococcus geothermalis 94986438 Dgeo_2341 DSM 11300 Streptococcus suis SC84 253752504 SSUSC84_1660 Bacillus coagulans 2-6 336113642 BCO26_0964 Wolbachia sp. wRi 225630846 WRi_011520 Streptococcus agalactiae 22537043 SAG0880 2603V/R Cyanothece sp. ATCC 51142 172037663 cce_2750 Desulfotalea psychrophila 51245946 DP2094 LSv54 Desulfotomaculum 333922988 Desca_0775 carboxydivorans CO 1 SRB Pseudomonas putida ND6 395447958 YSA_07990 Methanocella paludicola 282164389 MCP_1719 SANAE Nocardia cyriacigeorgica 379707337 NOCYR_1092 GUH-2 Leuconostoc gasicomitatum 300173680 LEGAS_1379 LMG 18811 Intrasporanglum calvum 317123422 Intca_0250 DSM 43043 Streptococcus pasteurianus 336064058 SGPB_0743 ATCC 43144 Thermus thermophilus HB27 46200056 TTC1754 Streptococcus thermophilus 116627818 STER_1034 LMD-9 Mycoplasma mycoides 42560815 MSC_0267 subsp. mycoides SC str. PG1 Burkholderia ambifaria 172060191 BamMC406_1136 MC40-6 Burkholderia pseudomallei 76818033 BURPS1710b_A1409 1710b Mycobacterium avium 104 118462619 MAV_1677 404488950 BLi01676 Shewanella frigidimarina 114563108 Sfri_1937 NCIMB 400 Staphylococcus aureus 379795466 SAMSHR1132_09420 subsp. aureus MSHR1132 Natronomonas pharaonis 76800930 NP0556A DSM 2160 Staphylococcus aureus 269202706 SAAV_1060 subsp. aureus ED98 Hirschia baltica ATCC 49814 254293978 Hbal_1616 Streptococcus thermophilus 55823000 str1049 CNRZ1066 Thermoplasma acidophilum 16082405 Ta1436 DSM 1728 Bacillus selenitireducens 297583900 Bsel_1604 MLS10 Mesorhizobium 337265442 Mesop_0913 opportunistum WSM2075 Sinorhizobium meliloti 1021 15966687 SMc03203 Salinispora tropica CNB-440 145594631 Strop_2099 Burkholderia mallei NCTC 126446955 BMA10247_A2300 10247 Desulfobulbus propionicus 320353605 Despr_1499 DSM 2032 Bdellovibrio bacteriovorus 42522358 Bd0779 HD100 Shewanella baltica OS155 126174436 Sbal_2220 Candidatus Phytoplasma 194246555 ATP_00154 mali Halobacillus halophilus DSM 386714109 HBHAL_2810 2266 Candidatus Phytoplasma 197294722 PAa_0688 australiense Geobacter bemidjiensis Bem 197116859 Gbem_0461 Staphylococcus 319892093 SPSINT_0804 pseudintermedius HKU10- 03 Mycobacterium bovis BCG 224990871 JTY_2509 str. Tokyo 172 Bordetella petril DSM 12804 163857822 Bpet3509 Listeria monocytogenes 406703830 LMOL312_1055 L312 Verrucosispora maris AB-18- 330468694 VAB18032_23695 032 Brucella canis ATCC 23365 161620588 BCAN_B0524 Brucella ovis ATCC 25840 148558405 BOV_A0457 Bacillus anthracis str. ‘Ames 47529478 GBAA_4182 Ancestor’ Caldicellulosiruptor 312622978 Calkro_1928 kronotskyensis 2002 Colwellia psychrerythraea 71279590 CPS_1584 34H Pseudovibrio sp. FO-BEG1 374331640 PSE_3294 Acetobacter pasteurianus 258542310 APA01_12160 IFO 3283-01 Mesorhizobium loti 13473767 mll4471 MAFF303099 Propionibacterium acnes 387504442 TIB1ST10_10615 6609 Pseudomonas putida BIRD-1 386011040 PPUBIRD1_1439 Cyanothece sp. PCC 7424 218437448 PCC7424_0443 Pyrobaculum calidifontis 126460011 Pcal_1403 JCM 11548 Stigmatella aurantiaca 31082197 STAUR_4668 DW4/3-1 Staphylococcus aureus 49483258 SAR1069 subsp. aureus MRSA252 Sanguibacter keddieli DSM 269795189 Sked_18850 10542 Pseudomonas aeruginosa 152984679 PSPA7_2992 PA7 Lactobacillus buchneri 331701106 Lbuc_0738 NRRL B-30929 Aerococcus urinae ACS-120- 326803934 HMPREF9243_1601 V-Col10a Bacillus atrophaeus 1942 311067976 BATR1942_05070 15609632 Rv2495c Staphylococcus aureus 156979418 SAHV_1087 subsp. aureus Mu3 Staphylococcus aureus 384549856 SAA6159_00951 subsp. aureus JKD6159 Candidatus Sulcia muelleri 293977962 DMIN_02500 DMIN Bacillus amyloliquefaciens 308173426 BAMF_1535 DSM 7 Anaeromyxobacter sp. K 197122441 AnaeK_2035 Isosphaera pallida ATCC 320101969 Isop_0416 43644 Maricaulis maris MCS10 114569257 Mmar10_0706 Anaeromyxobacter 220917208 A2cp1_2105 dehalogenans 2CP-1 Burkholderia cenocepacia 206559591 BCAL1214 J2315 Pseudomonas 330810244 PSEBR_a3380 brassicacearum subsp. brassicacearum NFM421 Synechococcus sp. RCC307 148242893 SynRCC307_1794 Staphylococcus aureus 15924085 SAV1095 subsp. aureus Mu50 Streptococcus thermophilus 55821074 stu1049 LMG 18311 Lactobacillus rhamnosus Lc 258539527 LC705_01336 705 Leuconostoc kimchii IMSNU 296110719 LKI_02940 11154 Mycoplasma gallisepticum 31544687 MGA_0162 str. R (low) Spirochaeta coccoides DSM 330837592 Spico_1653 17374 Brucella melitensis bv. 1 str. 17989091 BMEII0746 16M Shewanella baltica OS195 160875314 Sbal195_2201 Staphylococcus aureus 88194794 SAOUHSC_01042 subsp. aureus NCTC 8325 Nitratiruptor sp. SB155-2 152990678 NIS_0932 Leuconostoc citreum KM20 170016784 LCK_00426 Burkholderia sp. 383 78065833 Bcep18194_A4362 Bacillus pseudofirmus OF4 288553239 BpOF4_01070 Haliangium ochraceum DSM 262196421 Hoch_3235 14365 Variovorax paradoxus S110 239814019 Vapar_1012 Achromobacter xylosoxidans 311105813 AXYL_02631 A8 Ruegeria sp. TM1040 99082616 TM1040_2776 Sulfurovum sp. NBC37-1 152992561 SUN_0968 Geobacter sulfurreducens 39997750 GSU2656 PCA Chloroflexus aurantiacus J- 163846906 Caur_1333 10-fl Pseudoalteromonas 77360571 PSHAa1630 haloplanktis TAC125 Caulobacter crescentus 221234740 CCNA_01803 NA1000 Thermoproteus uzoniensis 327310925 TUZN_1029 768-20 Pseudomonas fluorescens 77459687 Pfl01_3465 Pf0-1 Lactobacillus casei ATCC 116494796 LSEI_1307 334 Prochlorococcus marinus 123967992 A9601_04551 str. AS9601 Lactobacillus casei str. 301066362 LCAZH_1301 Zhang Sphingobium japonicum 294011287 SJA_C1-13010 UT26S Cyanothece sp. PCC 8801 218246082 PCC8801_1231 Rhodothermus marinus 268316954 Rmar_1396 DSM 4252 Shewanella sp. W3-18-1 120598929 Sputw3181_2121 Nocardia farcinica IFM 54022990 nfa10230 10152 Bacillus subtilis subsp. 16078524 BSU14600 subtilis str. 168 Nocardioides sp. JS614 119718672 Noca_4453 Waddlia chondrophila WSU 297620707 wcw_0466 86-1044 Trichodesmium erythraeum 113475499 Tery_1831 IMS101 Bacillus tusciae DSM 2912 295694991 Btus_0312 Shewanella baltica OS223 217973404 Sbal223_2233 Frankia sp. Ccl3 86741182 Francci3_2486 Bacillus licheniformis ATCC 52080061 BL01618 14580 Rubrobacter xylanophilus 108805280 Rxyl_2478 DSM 9941 Conexibacter woesei DSM 284045846 Cwoe_4397 14684 Bacillus cereus B4264 218233546 BCB4264_A4073 Idiomarina loihiensis L2TR 56460779 IL1678 Bacillus clausii KSM-K16 56964183 ABC2418 Lactococcus lactis subsp. 125622951 llmg_0072 cremoris MG1363 Wolbachia endosymbiont of 42520975 WD1177 Drosophila melanogaster Caulobacter sp. K31 167645836 Caul_1872 Bacillus megaterium QM 294498115 BMQ_1348 B1551 Bacillus anthracis str. Sterne 49186879 BAS3881 Lactobacillus reuteri SD2112 338204230 HMPREF0538_21876 Halobacterium salinarum R1 169236792 OE4115F Streptococcus thermophilus 387909681 Y1U_C0873 MN-ZLW-002 Caulobacter crescentus 16125973 CC_1729 CB15 Symbiobacterium 51891551 STH413 thermophilum IAM 14863 Prochlorococcus marinus 33862493 PMT0220 str. MIT 9313 Shewanella pealeana ATCC 157962065 Spea_2244 700345 Mycobacterium sp. JDM601 333990060 JDM601_1420 Bacillus cereus 03BB102 225865952 BCA_4075 Agrobacterium sp. H13-3 332716272 AGROH133_11607 Bacillus cereus ATCC 10987 42783066 BCE_4019 Nostoc punctiforme PCC 186686433 Npun_F6414 73102 Bacillus anthracis str. A0248 229601593 BAA_4205 Mycobacterium smegmatis 399988989 MSMEI_4593 str. MC2 155 NC_018289 Shewanella amazonensis 119774847 Sama_1711 SB2B Bacillus cellulosilyticus DSM 317129370 Bcell_2666 2522 Synechococcus sp. WH 33865205 SYNW0671 8102 Brucella abortus S19 189022865 BAbS19_II06650 Acetobacter pasteurianus 384063023 APA32_12160 IFO 3283-32 Novosphingobium sp. PP1Y 334142272 PP1Y_AT28822 Listeria monocytogenes 217964853 LMHCC_1573 HCC23 Leuconostoc mesenteroides 116617851 LEUM_0739 subsp. mesenteroides ATCC 8293 Bacillus subtilis subsp. 350265764 GYO_1799 spizizenii TU-B-10 Halomicrobium mukohataei 257386742 Hmuk_0676 DSM 12286 Thermobifida fusca YX 72160586 Tfu_0182 Streptococcus gallolyticus 325978048 SGGBAA2069_c08480 subsp. gallolyticus ATCC BAA-2069 Burkholderia mallei NCTC 124383004 BMA10229_1320 10229 Prochlorococcus marinus 124025169 NATL1_04561 str. NATL1A Spirochaeta thermophila 307718514 STHERM_c08240 DSM 6192 Streptococcus suis ST3 330833463 SSUST3_1689 Synechococcus sp. CC9311 161349989 sync_0617 Streptococcus suis SS12 386582797 SSU12_1776 Burkholderia thailandensis 83716049 BTH_II2302 E264 Halomonas elongata DSM 307544962 HELO_2372 2581 Mycoplasma capricolum 83319741 MCAP_0227 subsp. capricolum ATCC 27343 Pseudomonas aeruginosa 218891777 PLES_30551 LESB58 Mycobacterium tuberculosis 15842023 MT2570 CDC1551 Alkaliphilus metalliredigens 150389069 Amet_1253 QYMF Sinorhizobium fredii 227823514 NGR_c29910 NGR234 Alteromonas macleodii str. 332141380 MADE_1009910 ‘Deep ecotype’ Geobacillus sp. C56-T3 297530773 GC56T3_2513 Leptospira borgpetersenii 116328021 LBL_1316 serovar Hardjo-bovis L550 Brucella microti CCM 4915 256015305 BMI_II520 Meiothermus ruber DSM 291296695 Mrub_2322 1279 Sphingopyxis alaskensis 103486815 Sala_1329 RB2256 Haloarcula marismortui 55380239 rrnB0198 ATCC 43049 Listeria monocytogenes 46907286 LMOf2365_1075 serotype 4b str. F2365 Thermosynechococcus 22298842 tll1299 elongatus BP-1 Mycoplasma genitallum G37 12045128 MG_272 Shewanella sp. ANA-3 117920573 Shewana3_2129 Bacillus weihenstephanensis 163941710 BcerKBAB4_3797 KBAB4 Shewanella putrefaciens CN- 146292985 Sputcn32_1887 32 Lactococcus lactis subsp. 116510883 LACR_0049 cremoris SK11 Burkholderia pseudomallei 53723289 BPSS2271 K96243 Synechococcus sp. PCC 170076743 SYNPCC7002_A0110 7002 Arthrobacter aurescens TC1 119963490 AAur_1523 Weissella koreensis KACC 339635177 WKK_06345 15510 Burkholderia mallei ATCC 53716063 BMAA2011 23344 Macrococcus caseolyticus 222150962 MCCL_0712 JCSC5402 Staphylococcus aureus 150393643 SaurJH1_1177 subsp. aureus JH1 Burkholderia cenocepacia 107022320 Bcen_0764 AU 1054 Meiothermus silvanus DSM 297565574 Mesil_1134 9946 Oceanobacillus iheyensis 23098869 OB1414 HTE831 Lactobacillus plantarum 380032903 lp_2152 WCFS1 Parabacteroides distasonis 150007338 BDI_0688 ATCC 8503 Staphylococcus 70726858 SH1857 haemolyticus JCSC1435 Lactobacillus fermentum IFO 184155614 LAF_1138 3956 Candidatus Amoebophilus 189501498 Aasi_0029 asiaticus 5a2 Catenulispora acidiphila 256395300 Caci_6169 DSM 44928 Bacillus amyloliquefaciens 154685877 RBAM_014440 FZB42 Mycobacterium bovis BCG 121638377 BCG_2515c str. Pasteur 1173P2 Staphylococcus aureus 212827071 MW0978 subsp. aureus MW2 Frankia sp. EAN1pec 158313432 Franean1_1595 Streptococcus equi subsp. 225870728 SEQ_1404 equi 4047 Gloeobacter violaceus PCC 37522138 gll2569 7421 Pseudomonas aeruginosa 116050196 PA14_35500 UCBPP-PA14 Prochlorococcus marinus 157412817 P9215_04811 str. MIT 9215 Thermoplasma volcanium 13540931 TVN0100 GSS1 Bacillus megaterium DSM 295703464 BMD_1328 319 Anabaena variabilis ATCC 75909383 Ava_3176 29413 Paenlbacillus polymyxa 308069293 PPE_02530 E681 Chlamydophila abortus 62185091 CAB462 S26/3 Geobacter uraniireducens 148264937 Gura_2898 Rf4 Lactobacillus casei BL23 191638311 LCABL_15380 Streptococcus agalactiae 76787510 SAK_003 A909 Pseudomonas entomophila 104782859 PSEEN3855 L48 Halorubrum lacusprofundi 222478580 Hlac_0141 ATCC 49239 Geobacillus kaustophilus 56419595 GK1060 HTA426 Staphylococcus aureus 87161817 SAUSA300_0995 subsp. aureus USA300_FPR3757 Azospirillum sp. B510 288958360 AZL_015190 Chlamydophila psittaci 384452547 CPS0D_0522 08DC60 Lactobacillus rhamnosus 258508317 LGG_01322 GG NC_013198 Staphylococcus aureus 82750705 SAB0961 RF122 cyanobacterium UCYN-A 284928764 UCYN_01790 Zymomonas mobilis subsp. 338707699 Zymop_0708 pomaceae ATCC 29192 Burkholderia cenocepacia 170732568 Bcenmc03_1218 MC0-3 Thermus scotoductus SA-01 320449329 TSC_c02360 Candidatus Protochlamydia 46447365 pc1731 amoebophila UWE25 Bacillus cereus biovar 301055461 BACI_c39270 anthracis str. CI Burkholderia cenocepacia 116689267 Bcen2424_1245 HI2424 Oenococcus oeni PSU-1 116490426 OEOE_0330 Listeria monocytogenes 08- 284801386 LM5578_1137 5578 Alteromonas sp. SN2 333893050 ambt_07975 Shewanella sp. MR-4 113970363 Shewmr4_2026 Streptomyces avermitilis MA- 29830907 SAV_4364 4680 Mycoplasma hyorhinis HUB-1 304373301 MHR_0515 uncultured methanogenic 147919054 RRC124 archaeon RC-I Staphylococcus aureus 15926680 SA0945 subsp. aureus N315 Prochlorococcus marinus 33860962 PMM0405 subsp. pastoris str. CCMP1986 Bacteroides fragilis YCH46 53712911 BF1621 Thermobispora bispora DSM 296271281 Tbis_3330 43833 Amycolatopsis mediterranei 300789393 AMED_7569 U32 Paenibacillus sp. Y412MC10 261406247 GYMC10_2403 Erysipelothrix rhusiopathiae 336065680 ERH_0440 str. Fujisawa Sulfurimonas autotrophica 307720480 Saut_0559 DSM 16294 Acaryochloris marina 158336703 AM1_3571 MBIC11017 Lactobacillus plantarum 254556970 JDM1_1803 JDM1 Streptococcus equi subsp. 195978329 Sez_1220 zooepidemicus MGCS10565 Bacillus thuringiensis 296504464 BMB171_C3634 BMB171 Mycobacterium avium 41408405 MAP2307c subsp. paratuberculosis K- 10 Mycoplasma fermentans 308190216 MFE_06880 JER Leptospira interrogans 24214708 LA_2008 serovar Lai str. 56601 Brucella melitensis biovar 83269596 BAB2_0713 Abortus 2308 Rhodococcus jostii RHA1 111020308 RHA1_ro03319 Ramlibacter tataouinensis 337278683 Rta_10500 TTB310 Mycoplasma fermentans 319777549 MfeM64YM_0828 M64 Ochrobactrum anthropi 153010874 Oant_3553 ATCC 49188 Erythrobacter litoralis 85373860 ELI_05165 HTCC2594 Bacillus subtilis BSn5 321315219 BSn5_19375 Pseudomonas putida GB-1 167034959 PputGB1_3964 Prochlorococcus marinus 123965701 P9515_04661 str. MIT 9515 Bacillus cereus Q1 222097419 BCQ_3759 Cyanothece sp. PCC 7822 307154646 Cyan7822_4864 Anoxybacillus flavithermus 212639718 Aflv_1892 WK1 Bacillus cereus AH187 217961456 BCAH187_A4089 Burkholderia ambifaria 115351178 Bamb_1125 AMMD Streptococcus uberis 0140J 222153239 SUB1101 Sinorhizobium medicae 150398026 Smed_2828 WSM419 Halogeometricum 313125080 Hbor_02950 borinquense DSM 11551 Mycobacterium vanbaalenii 120405038 Mvan_4084 PYR-1 Halalkalicoccus jeotgali B3 300709397 HacjB3_00130 Lysinibacillus sphaericus C3- 169826945 Bsph_1365 41 Exiguobacterium sibiricum 172058018 Exlg_2009 255-15 Mycoplasma mobile 163K 47459416 MMOB5810 Streptococcus suis P1/7 253754330 SSU1635 Geobacillus sp. Y4.1MC1 312111829 GY4MC1_2839 Anaeromyxobacter sp. 153004857 Anae109_1995 Fw109-5 Simkania negevensis Z 338732264 SNE_A03690 Micromonospora sp. L5 315504011 ML5_3231 Listeria monocytogenes 16803094 Imo1054 EGD-e Cyanothece sp. PCC 8802 257059131 Cyan8802_1261 Prochlorococcus marinus 159902944 P9211_04031 str. MIT 9211 Carnobacterium sp. 17-4 328957243 CAR_c09200 Zymomonas mobilis subsp. 260752983 Za10_0745 mobilis NCIMB 11163 Leptospira borgpetersenil 116331526 LBJ_1968 serovar Hardjo-bovis JB197 Saccharopolyspora 134101994 SACE_5544 erythraea NRRL 2338 Pusillimonas sp. T7-7 332284091 PT7_0838 Pediococcus pentosaceus 116493499 PEPE_1771 ATCC 25745 Shewanella piezotolerans 212635403 swp_2604 WP3 Streptosporangium roseum 271970150 Sros_8972 DSM 43021 Lactobacillus reuteri JCM 184153265 LAR_0610 1112 Paenibacillus polymyxa SC2 310642335 PPSC2_c2887 ‘Nostoc azollae’ 0708 298490612 Aazo_1439 Rhodoferax ferrireducens 89902316 Rfer_3552 T118 Streptomyces griseus 182437543 SGR_3750 subsp. griseus NBRC 13350 Pyrobaculum aerophilum str. 18313491 PAE2648 IM2 Mycobacterium smegmatis 118470681 MSMEG_4710 str. MC2 155 NC_008596 Mycobacterium abscessus 169627994 MAB_0895c ATCC 19977 Thermoanaerobacter 20806714 TTE0188 tengcongensis MB4 Cyanothece sp. PCC 7425 220910325 Cyan7425_4977 Brevibacillus brevis NBRC 226312883 BBR47_32960 100599 Salinispora arenicola CNS- 159037838 Sare_2240 205 Listeria monocytogenes 08- 284994528 LM5923_1091 5923 Sphaerobacter thermophilus 269929375 Sthe_3475 DSM 20745 Mycobacterium tuberculosis 148823692 TBFG_12517 F11 Mycoplasma 54020221 mhp503 hyopneumoniae 232 Marinithermus 328950233 Marky_0708 hydrothermalis DSM 14884 Mycoplasma mycoides 331703290 MLC_2700 subsp. capri LC str. 95010 Listeria welshimeri serovar 116872448 lwe1030 6b str. SLCC5334 Glaciecola sp. 4H-3-7 + YE-5 332306592 Glaag_2230 Geobacter metallireducens 404497364 Gmet_2511 GS-15 Nostoc sp. PCC 7120 17231098 alr3606 Mycoplasma 71893854 MHJ_0503 hyopneumoniae J Propionibacterium acnes 50843531 PPA2092 KPA171202 Picrophilus torridus DSM 48477619 PTO0547 9790 Staphylococcus aureus 151221173 NWMN_0961 subsp. aureus str. Newman 319766073 GYMC52_0960 Geobacillus sp. Y412MC52 Ferrimonas balearica DSM 308050066 Fbal_2356 9799 Listeria monocytogenes 226223672 Lm4b_01074 serotype 4b str. CLIP 80459 Sinorhizobium meliloti AK83 334317692 Sinme_2990 Prochlorococcus marinus 72383575 PMN2A_1739 str. NATL2A Sphingobacterium sp. 21 326799941 Sph21_2539 Pseudomonas putida W619 170722907 PputW619_3744

TABLE 5 IpdV Genes Genome Gene ID# Locus Tag Arcobacter sp. L 384172498 ABLL_1683 Listeria monocytogenes SLCC2479 405758055 LMOSLCC2479_1068 Bacillus coagulans 36D1 3477506611 Bcoa_0220 Lactococcus lactis subsp. 389853268 LLNZ_00350 cremoris NZ9000 Staphylococcus aureus subsp. 384869629 SAT0131_01134 aureus T0131 Mycoplasma bovis HB0801 392429697 Mbov_0106 Sulfobacillus acidophilus TPY 339629488 TPY_3236 Listeria ivanovii subsp. ivanovii 347548456 LIV_1000 PAM 55 Listeria monocytogenes M7 386026372 LMM7_1085 Bacillus subtills subsp. subtills 384175197 I33_1641 str. RO-NN-1 Listeria monocytogenes 404413142 LMOSLCC7179_1036 SLCC7179 Sinorhizobium fredii HH103 378827586 SFHH103_03001 Sulfobacillus acidophilus DSM 379007394 Sulac_1677 10332 Staphylococcus aureus subsp. 379020803 M013TW_1028 aureus M013 Haloferax mediterranel ATCC 389848367 HFX_2955 33500 Enterococcus hirae ATCC 9790 392989733 EHR_12850 Pseudomonas fluorescens F113 378950261 PSF113_2343 Staphylococcus aureus subsp. 385781324 MS7_1053 aureus 11819-97 Staphylococcus aureus subsp. 387780209 SARLGA251_10090 aureus LGA251 Staphylococcus aureus subsp. 379014289 SAVC_04640 aureus VC40 Haloquadratum walsbyi C23 385802475 Hqrw_1115 Staphylococcus aureus subsp. 384547282 SAOV_1040 aureus ED133 Mycoplasma bovis Hubei-1 339320627 MMB_0100 Methanocella conradii HZ254 383320310 Mtc_1893 Sinorhizobium meliloti SM11 384537527 SM11_chr3107 Desulfosporosinus acidiphilus 392426986 Desaci_3767 SJ4 Pyrobaculum sp. 1860 374327310 P186_1854 Paenibacillus polymyxa M1 386041285 PPM_2595 Bacillus amyloliquefaciens LL3 384163957 LL3_01567 Listeria monocytogenes J0161 386046717 LMOG_00665 Bacillus megaterium WSH-002 384048054 BMWSH_3881 Lactobacillus casei BD-II 385823212 LCBD_1516 Listeria monocytogenes 404410300 LMOSLCC5850_1061 SLCC5850 Melissococcus plutonius DAT56 379727535 MPD5_0995 Pyrobaculum oguniense TE7 379004031 Pogu_1069 Amycolatopsis mediterranei 384147341 RAM_11050 S699 NC_017186 Bacillus cereus NC7401 375285964 BCN_3870 Listeria monocytogenes 405755134 LMOSLCC2540_1055 SLCC2540 Staphylococcus aureus subsp. 386728776 ST398NM01_1093 aureus 71193 Brucella suis VBI22 376278495 BSVBI22_B0521 Listeria monocytogenes L99 386007779 Imo4a_1064 Exiguobacterium antarcticum 407477696 Eab7_1857 B7 Sulfolobus solfataricus 98/2 384433026 Ssol_0504 Listeria monocytogenes 404407511 LMOSLCC2376_1028 SLCC2376 Ignavibacterium album JCM 385811557 IALB_2982 16511 Mycobacterium tuberculosis 386006142 MRGA423_20730 RGTB423 Staphylococcus lugdunensis 385784711 SLUG_17740 N920143 Bacillus amyloliquefaciens 384265012 BANAU_1382 subsp. plantarum YAU B9601-Y2 Corynebacterium variabile 340793902 CVAR_0939 DSM 44702 Anaerobaculum mobile DSM 392407596 Anamo_1266 13181 Haloarcula hispanica ATCC 344210514 HAH_0213 33960 Lactobacillus rhamnosus GG 385827989 LRHM_1269 NC_017482 Geobacillus thermoleovorans 375008019 GTCCBUS3UF5_12380 CCB_US3_UF5 Brucella melitensis M5-90 384213037 BM590_B0499 Staphylococcus 386319626 SPSE_1695 pseudintermedius ED99 Lactobacillus casei LC2W 385820011 LC2W_1481 Bacillus amyloliquefaciens 375362109 BACAU_1419 subsp. plantarum CAU B946 Staphylococcus aureus subsp. 384864323 ECTR2_951 aureus ECT-R 2 Listeria monocytogenes 404286469 LMOSLCC2482_1102 serotype 7 str. SLCC2482 Staphylococcus aureus subsp. 384868022 HMPREF0772_12136 aureus TCH60 Lactobacillus rhamnosus ATCC 385835177 LRHK_1313 8530 Pseudomonas aeruginosa 386066188 NCGM2_3259 NCGM2.S1 Bacillus anthracis str. H9401 386737859 H9401_3986 Bacillus amyloliquefaciens 384159555 BAMTA208_09840 TA208 Pseudomonas fluorescens A506 387894502 PflA506_3340 Sinorhizobium meliloti BL225C 384530817 SinmeB_2765 Bacillus sp. JS 386758177 MY9_1600 Listeria monocytogenes 405752277 LMOSLCC2378_1073 SLCC2378 Listeria monocytogenes Finland 386053326 LMLG_2332 1998 Arcobacter butzleri ED-1 384156087 ABED_1375 Brucella melitensis NI 384446663 BMNI_II0492 Brachyspira pilosicoli B2904 404476148 B2904_orf1496 Alicyclobacillus acidocaldarius 384134350 TC41_0585 subsp. acidocaldarius Tc-4-1 Thermoanaerobacter wiegelii 345016728 Thewi_0296 Rt8.B1 Lactobacillus buchneri CD034 406026603 LBUCD034_0784 Brucella canis HSK A52141 376276772 BCA52141_II0438 Bacillus amyloliquefaciens XH7 384168609 BAXH7_02007 Pseudomonas aeruginosa M18 386058853 PAM18_2790 Enterococcus faecium Aus0004 383328410 EFAU004_01091 halophilic archaeon DL31 345006004 Halar_2852 Tetragenococcus halophilus 352517700 TEH_15260 NBRC 12172 Staphylococcus aureus 04-02981 387150238 SA2981_1053 Thermoanaerobacterium 390935443 Tsac_2347 saccharolyticum JW/SL-YS485 Staphylococcus aureus subsp. 386830632 SAEMRSA15_09260 aureus HO 5096 0412 Paenibacillus mucilaginosus 379723634 PM3016_5959 3016 Pseudomonas putida S16 339488701 PPS_3808 Lactobacillus salivarius 385839944 HN6_00133 CECT 5713 Marinobacter adhaerens HP15 385329806 HP15_65 Burkholderia cepacia GG4 402566976 GEM_2214 Brucella melitensis M28 384410138 BM28_B0500 Listeria monocytogenes 10403S 386043381 LMRG_00517 Sphingobium sp. SYK-6 347527353 SLG_09680 Enterococcus faecium DO 389868411 HMPREF0351_11228 Bacillus thuringiensis serovar 384181786 YBT020_19510 finitimus YBT-020 Bacillus cereus F837/76 376267866 bcf_19720 Thermoproteus tenax Kra 1 352681565 TTX_0334 Brachyspira intermedia PWS/A 384208204 Bint_0715 Paenibacillus mucilaginosus 386726383 B2K_30290 K02 Enterococcus faecalis 62 384518457 EF62_1807 Listeria monocytogenes ATCC 405749412 LMOATCC19117_1077 19117 Listeria monocytogenes FSL R2- 386049983 LMKG_01867 561 Natrinema sp. J7-2 397771794 NJ7G_0008 Pseudomonas putida DOT-T1E 397697837 T1E-5102 Burkholderia pseudomallei 386866104 BP1026B_II2445 1026b Staphylococcus aureus subsp. 384861691 SAA6008_01051 aureus str. JKD6008 Listeria monocytogenes 386731810 MUO_05550 07PF0776 Solibacillus silvestris StLB046 393201878 SSIL_3151 Paenibacillus terrae HPL-003 374323890 HPL003_20290 Staphylococcus aureus subsp. 387602369 SAPIG1093 aureus S0385 Staphylococcus aureus subsp. 387142708 SATW20_10910 aureus TW20 Brucella pinnipedialis B2/94 340792255 BPI_II508 384223057 BS1330_II0522 Streptococcus parasanguinis 387879561 Spaf_1060 FW213 Sinorhizobium fredii USDA 257 398355224 USDA257_c54080 Enterococcus faecalis D32 397699745 EFD32_1168 Bacillus amyloliquefaciens Y2 387897997 MUS_1556 Bacillus anthracis str. CDC 684 227816743 BAMEG_4222 Lactobacillus sakei subsp. sakei 81428692 LSA1082 23K Bacillus halodurans C-125 15615215 BH2652 Paenibacillus sp. JDR-2 251796662 Pjdr2_2653 Listeria seeligeri serovar 289434316 Ise_0949 1/2b str. SLCC3954 Pseudomonas fluorescens 229591399 PFLU3967 SBW25 Halopiger xanaduensis SH-6 336252524 Halxa_1118 Pseudomonas fluorescens Pf-5 70729899 PFL_2531 Bacillus subtilis subsp. 305674188 BSUW23_07515 spizizenii str. W23 Bradyrhizobium japonicum 27381445 blr6334 USDA 110 Lactobacillus salivarius 90961138 LSL_0156 UCC118 Paenibacillus mucilaginosus 337750575 KNP414_06346 KNP414 Haloferax volcanii DS2 292657069 HVO_2961 Brucella melitensis 225686319 BMEA_B0500 ATCC 23457 Lactobacillus reuteri 148543866 Lreu_0634 DSM 20016 Bacillus pumilus SAFR-032 157692139 BPUM_1358 Bacillus cereus G9842 218899125 BCG9842_B1168 Arthrobacter arilaitensis Re117 308177811 AARI_20270 Pseudomonas putida KT2440 26991093 PP_4404 Haloterrigena turkmenica DSM 284166850 Htur_3594 5511 Rothia dentocariosa ATCC 311111675 HMPREF0733_10005 17931 Enterococcus faecalis V583 29375923 EF1356 Staphylococcus epidermidis 57866609 SERP0683 RP62A Staphylococcus aureus subsp. 148267589 SaurJH9_1156 aureus JH9 Staphylococcus aureus subsp. 57651705 SACOL1105 aureus COL Staphylococcus epidermidis 27467712 SE0794 ATCC 12228 Buchnera aphidicola (Cinara 336233239 BCTU_137 tujafilina) Brucella suis 1330 23500274 BRA0527 Bacillus cereus AH820 218905100 BCAH820_3984 Thermaerobacter marianensis 317122503 Tmar_1670 DSM 12885 Desulfovibrio magneticus RS-1 239908133 DMR_34970 Burkholderia pseudomallei 126455671 BURPS1106A_A3064 1106a Geobacillus sp. Y412MC61 261419259 GYMC61_1835 Mesorhizobium ciceri biovar 319780623 Mesci_0884 biserrulae WSM1271 Calditerrivibrio nitroreducens 313672838 Calni_0875 DSM 19672 Wigglesworthia glossinidia 32491070 WGLp321 endosymbiont of Glossina brevialpis Exiguobacterium sp. AT1b 229918501 EAT1b_2788 Flexistipes sinusarabici 336323720 Flexsi_1469 DSM 4947 Lactobacillus plantarum subsp. 308180957 LPST_C1775 plantarum ST-III Bacillus anthracis str. Ames 30264041 BA_4181 Halobacterium sp. NRC-1 15791043 VNG2220G Bacillus cereus E33L 52141520 BCZK3728 Geobacillus thermodenitrificans 138894595 GTNG_0925 NG80-2 Burkholderia glumae BGR1 238023735 bglu_2g02670 Alicycliphilus denitrificans BC 319761350 Alide_0631 Staphylococcus aureus subsp. 161509279 USA300HOU_1039 aureus USA300_TCH1516 Bacillus thuringiensis str. 118479181 BALH_3592 Al Hakam Bacillus thuringiensis serovar 384188033 CT43_CH3975 chinensis CT-43 Brucella abortus A13334 376271254 BAA13334_II01340 Bacillus thuringiensis serovar 49478876 BT9727_3712 konkukian str. 97-27 Alicycliphilus denitrificans K601 330823225 Allde2_0596 Baumannia cicadellinicola 94676976 BCI_0510 str. Hc (Homalodisca coagulata) Geobacillus thermoglucosidasius 336236212 Geoth_2860 C56-YS93 Alicyclobacillus acidocaldarius 258510468 Aaci_0454 subsp. acidocaldarius DSM 446 Rothia mucilaginosa DY-18 283457790 RMDY18_07290 Burkholderia pseudomallei 668 126442365 BURPS668_A3190 Jonesia denitrificans DSM 256832700 Jden_1475 20603 Burkholderia gladioli BSR3 330819433 bgla_2g03070 Staphylococcus aureus subsp. 49485934 SAS1031 aureus MSSA476 Sphingomonas wittichii RW1 148553702 Swit_0779 Staphylococcus lugdunensis 289551092 SLGD_01779 HKU09-01 Staphylococcus saprophyticus 73663002 SSP1693 subsp. saprophyticus ATCC 15305 Natrialba magadii ATCC 43099 289581337 Nmag_1665 Brucella suis ATCC 23445 163844685 BSUIS_B0522 Listeria innocua Clip11262 16800116 lin1047 Bacillus cereus ATCC 14579 30022058 BC3970 Pseudomonas putida F1 148546690 Pput_1450 Brucella abortus bv. 1 str. 9-941 62317612 BruAb2_0697 Burkholderia mallei SAVP1 121597939 BMASAVP1_1033 Pseudomonas aeruginosa DK2 392984201 PADK2_14025 Brevundimonas subvibrioides 302384433 Bresu_3327 ATCC 15264 Sphingobium chlorophenolicum 334342794 Sphch_3288 L-1 Thermoanaerobacter sp. X513 307724064 Thet_0899 Staphylococcus carnosus subsp. 224476211 Sca_0722 carnosus TM300 Lactobacillus fermentum CECT 385812452 LC40_0740 5716 Geobacillus sp. WCH70 239826460 GWCH70_0955 Bacillus coagulans 2-6 336113643 BCO26_0965 Kocuria rhizophila DC2201 184201087 KRH_11410 Pseudomonas putida ND6 395447957 YSA_07988 Methanocella paludicola 282164390 MCP_1720 SANAE Leuconostoc gasicomitatum 300173679 LEGAS_1378 LMG 18811 Burkholderia ambifaria 172060192 BamMC406_1137 MC40-6 Burkholderia pseudomallei 76818035 BURPS1710b_A1408 1710b Mycoplasma agalactiae 291320035 MAGa1040 404488951 BLi01677 Staphylococcus aureus subsp. 379795467 SAMSHR1132_09430 aureus MSHR1132 Natronomonas pharaonis DSM 76800707 NP0104A 2160 Staphylococcus aureus subsp. 269202707 SAAV_1061 aureus ED98 Hirschia baltica ATCC 49814 254293979 Hbal_6117 Thermoplasma acidophilum 16082404 Ta1435 DSM 1728 Bacillus selenitireducens MLS10 297583901 Bsel_1605 Mesorhizobium opportunistum 337265443 Mesop_0914 WSM2075 Sinorhizobium meliloti 1021 15966688 SMc03204 Burkholderia mallei NCTC 126447169 BMA10247_A2299 10247 Halobacillus halophilus DSM 386714110 HBHAL_2811 2266 Staphylococcus 319892094 SPSINT_0805 pseudintermedius HKU10-03 Bordetella petrii DSM 12804 163857821 Bpet3508 Listeria monocytogenes L312 406703831 LMOL312_1056 Brucella canis ATCC 23365 161620589 BCAN_B0525 Brucella ovis ATCC 25840 148558309 BOV_A0459 Bacillus anthracis str. ‘Ames 47529477 GBAA_4181 Ancestor’ Mesorhizobium loti 13473766 mll4470 MAFF303099 Pseudomonas putida BIRD-1 386011039 PPUBIRD1_1438 Pyrobaculum calidifontis JCM 126460010 Pcal_1402 11548 Candidatus Biochmannia 71891939 BPEN_156 pennsylvanicus str. BPEN Staphylococcus aureus subsp. 49483259 SAR1070 aureus MRSA252 Mycoplasma agalactiae PG2 148377364 MAG_0960 Pseudomonas aeruginosa PA7 152989284 PSPA7_2991 Lactobacillus buchneri NRRL 331701107 Lbuc_0739 B-30929 Aerococcus urinae ACS- 326803933 HMPREF9243_1600 120-V-Col10a Bacillus atrophaeus 1942 311067977 BATR1942_05075 Thermoanaerobacter sp. X514 167040661 Teth514_2038 Staphylococcus aureus subsp. 156979419 SAHV_1088 aureus Mu3 Staphylococcus aureus subsp. 384549857 SAA6159_00952 aureus JKD6159 Bacillus amyloliquefaciens 308173427 BAMF_1536 DSM 7 Maricaulis maris MCS10 114569260 Mmar10_0709 Thermoanaerobacter brockii 320115619 Thebr_0807 subsp. finnii Ako-1 Burkholderia cenocepacia J2315 206559592 BCAL1215 Pseudomonas brassicacearum 330810245 PSEBR_a3381 subsp. brassicacearum NFM421 Staphylococcus aureus subsp. 15924086 SAV1096 aureus Mu50 Lactobacillus rhamnosus Lc 705 258539528 LC705_01337 Spirochaeta coccoides DSM 330837591 Spico_1652 17374 Brucella melitensis bv. 17989090 BMEII0745 1 str. 16M Staphylococcus aureus subsp. 88194795 SAOUHSC_01043 aureus NCTC 8325 Leuconostoc citreum KM20 170016785 LCK_00427 Burkholderia sp. 383 78065834 Bcep18194_A4363 Prevotella melaninogenica 302346454 HMPREF0659_A6708 ATCC 25845 Bacillus pseudofirmus OF4 288553238 BpOF4_01065 Achromobacter xylosoxidans A8 311105814 AXYL_02632 Ruegeria sp. TM1040 99082615 TM1040_2775 Metallosphaera sedula DSM 146304009 Msed_1241 5348 Borrelia garinii PBi 51598980 BG0750 Thermoproteus uzoniensis 327310926 TUZN_1030 768-20 Pseudomonas fluorescens Pf0-1 77459688 Pfl01_3466 Lactobacillus casei ATCC 334 116494797 LSEI_1308 Lactobacillus casei str. Zhang 301066363 LCAZH_1302 Bacillus subtilis subsp. 16078525 BSU14610 subtilis str. 168 Bacillus tusciae DSM 2912 295694992 Btus_0313 Bacillus licheniformis ATCC 52080062 BL01619 14580 Borrelia turicatae 91E135 119953505 BT0728 Slackia heliotrinireducens DSM 257064081 Shel_13830 20476 Acidilobus saccharovorans 302348861 ASAC_1063 345-15 Conexibacter woesel DSM 284045845 Cwoe_4396 14684 Bacillus cereus B4264 218233948 BCB4264_A4072 Prevotella denticola F0289 327314405 HMPREF9137_2189 Bacillus clausii KSM-K16 56964182 ABC2417 Lactococcus lactis subsp. 125622950 llmg_0071 cremoris MG1363 Dictyoglomus turgidum DSM 217966916 Dtur_0515 6724 Methanococcus voltae A3 297619150 Mvol_0623 Caulobacter sp. K31 167645837 Caul_1873 Enterococcus faecalis OG1RF 384513108 OG1RF_11144 Bacillus megaterium QM B1551 294498116 BMQ_1349 Bacillus anthracis str. Sterne 49186878 BAS3880 Bacillus cytotoxicus NVH 152976383 Bcer98_2671 391-98 Lactobacillus reuteri SD2112 338204231 HMPREF0538_21877 Halobacterium salinarum R1 169236793 OE4116F Symbiobacterium thermophilum 51891552 STH414 IAM 14863 Chlorobium phaeobacteroides 189499997 Cphamn1_1045 BS1 Bacillus cereus 03BB102 225865951 BCA_4074 Bacillus cereus ATCC 10987 42783065 BCE_4018 Vulcanisaeta moutnovskia 325968467 VMUT_0947 768-28 Bacillus anthracis str. A0248 229602624 BAA_4204 Bacillus cellulosilyticus DSM 317129369 Bcell_2665 2522 Brucella abortus S19 189022864 BAbS19_II06640 Mycobacterium leprae TN 15828281 ML2387 Haloquadratum walsbyi DSM 110667061 HQ1085A 16790 Listeria monocytogenes HCC23 217964852 LMHCC_1572 Bacillus subtilis subsp. spizizenii 350265765 GYO_1800 TU-B-10 Halomicrobium mukohataei 257387048 Hmuk_0985 DSM 12286 Burkholderia mallei NCTC 124381655 BMA10229_1319 10229 Burkholderia thailandensis 83717960 BTH_II2301 E264 Pseudomonas aeruginosa 218891776 PLES_30541 LESB58 Sinorhizobium fredii NGR234 227823515 NGR_c29920 Geobaclllus sp. C56-T3 297530772 GC56T3_2512 Brucella microti CCM 4915 256015306 BMI_II521 Haloarcula marismortui ATCC 55379543 rrnAC2953 43049 Listeria monocytogenes serotype 46907287 LMOf2365_1076 4b str. F2365 Bacillus weihenstephanensis 163941709 BcerKBAB4_3796 KBAB4 Burkholderia pseudomallei 53723288 BPSS2270 K96243 Brachyspira murdochii DSM 296125823 Bmur_0777 12563 Thermoanaerobacterium 304317378 Tthe_1955 thermosaccharolyticum DSM 571 Mycobacterium ulcerans 118617748 MUL_2214 Agy99 Burkholderia mallei 53716068 BMAA2010 ATCC 23344 Macrococcus caseolyticus 222150963 MCCL_0713 JCSC5402 Desulfovibrio vulgaris str. 218886000 DvMF_0898 ‘Miyazaki F’ Staphylococcus aureus subsp. 150393644 SaurJH1_1178 aureus JH1 Burkholderia cenocepacia AU 107022321 Bcen_0765 1054 Mycobacterium marinum M 183980809 MMAR_0785 Oceanobacillus iheyensis 23098870 OB1415 HTE831 Lactobacillus plantarum 380032902 lp_2151 WCFS1 Staphylococcus haemolyticus 70726857 SH1856 JCSC1435 Lactobacillus fermentum IFO 184155613 LAF_1137 3956 Bacillus amyloliquefaciens 154685878 RBAM_014450 FZB42 Staphylococcus aureus subsp. 21282708 MW0979 aureus MW2 Pseudomonas aeruginosa 116050197 PA14_35490 UCBPP-PA14 Thermoplasma volcanium GSS1 13540930 TVN0099 Sulfolobus solfataricus P2 15899410 SSO2689 Bacillus megaterium DSM 319 295703465 BMD_1329 Paembacillus polymyxa E681 308069292 PPE_02529 Lactobacillus casei BL23 191638312 LCABL_15390 Pseudomonas entomophila L48 104782860 PSEEN3856 Halorubrum lacusprofundi 222478581 Hlac_0142 ATCC 49239 Geobacillus kaustophilus 56419596 GK1061 HTA426 Staphylococcus aureus subsp. 87161349 SAUSA300_0996 aureus USA300_FPR3757 actobacillus rhamnosus GG 258508318 LGG_01323 NC_013198 Staphylococcus aureus RF122 82750706 SAB0962 Burkholderia cenocepacla 170732569 Bcenmc03_1219 MC0-3 Thermoanaerobacter 167037200 Teth39_0785 pseudethanolicus ATCC 33223 Bacillus cereus biovar 301055460 BACI_c39260 anthracis str. CI Burkholderia cenocepacia 116689268 Bcen2424_1246 HI2424 Oenococcus oeni PSU-1 116490427 OEOE_0331 Listeria monocytogenes 08-5578 284801387 LM5578_1138 Clostridium tetani E88 28211667 CTC02047 Thermoanaerobacterium 333896751 Thexy_0914 xylanolyticum LX-11 Brachyspira pilosicoli 300869836 BP951000_0199 95/1000 uncultured 147919055 RRC122 methanogenic archaeon RC-I Staphylococcus aureus subsp. 15926681 SA0946 aureus N315 Erysipelothrix rhusiopathiae 336065681 ERH_0441 str. Fujisawa Mycobacterium leprae Br4923 221230758 MLBr_02387 Lactobacillus plantarum JDM1 254556969 JDM1_1802 Pyrobaculum arsenaticum DSM 145591423 Pars_1205 13514 Mycoplasma bovis PG45 313678233 MBOVPG45_0108 Bacillus thuringiensis 296504463 BMB171_C3633 BMB171 Arcobacter butzleri RM4018 157737715 Abu_1474 Mycobacterium avium subsp. 41410054 MAP3956 paratuberculosis K-10 Brucella melitensis biovar 83269595 BAB2_0712 Abortus 2308 Ramlibacter tataouinensis 337278684 Rta_10510 TTB310 Ochrobactrum anthropi ATCC 153010875 Oant 3554 49188 Bacillus subtilis BSn5 321315220 BSn5_19380 Pseudomonas putida GB-1 167034960 PputGB1_3965 Bacillus cereus Q1 222097418 BCQ_3758 Arcobacter nitrofigilis 296274130 Arnit_2606 DSM 7299 Anoxybacillus flavithermus 212639717 Aflv_1891 WK1 Lactobacillus brevis ATCC 116334009 LVIS_1407 367 Bacillus cereus AH187 217961455 BCAH187_A4088 Burkholderia ambifaria AMMD 115351179 Bamb_1126 Sinorhizobium medicae 150398027 Smed_2829 WSM419 Halogeometricum borinquense 313125079 Hbor_02940 DSM 11551 Halalkalicoccus jeotgali B3 300709398 HacjB3_00135 Lysinibacilius sphaericus C3-41 169826946 Bsph_1366 Exiguobacterium sibiricum 172058017 Exig_2008 Geobacillus sp. Y4.1MC1 312111828 GY4MC1_2838 255-15 Anaeromyxobacter sp. Fw109-5 153004860 Anae109_1998 Listeria monocytogenes EGD-e 16803095 lmo1055 Carnobacterium sp. 17-4 328957244 CAR_c09210 Campylobacter concisus 13826 157164634 CCC13826_0588 Pusillimonas sp. T7-7 332284092 PT7_0839 Sulfolobus acidocaldarius 70607437 Saci_1708 DSM 639 Pediococcus pentosaceus ATCC 116493498 PEPE_1170 25745 Lactobacillus reuteri JCM 184153266 LAR_0611 1112 Paenibacillus polymyxa SC2 310642334 PPSC2_c2886 Pyrobaculum aerophilum str. 18313492 PAE2649 IM2 Halorhabdus utahensis DSM 257052876 Huta_1805 12940 Thermoanaerobacter 20806617 TTE0088 tengcongensis MB4 Brevibacillus brevis NBRC 226312882 BBR47_32950 100599 Brachyspira hyodysenteriae 225621390 BHWA1_02490 WA1 Listeria monocytogenes 08-5923 284994529 LM5923_1092 Listeria welshimeri serovar 116872449 lwe1031 6b str. SLCC5334 Picrophilus torridus DSM 48477618 PTO0546 9790 Staphylococcus aureus subsp. 151221174 NWMN_0962 aureus str. Newman Geobacillus sp. Y412MC52 319766074 GYMC52_0961 Listeria monocytogenes 226223673 Lm4b_01075 serotype 4b str. CLIP 80459 Sinorhizobium meliloti AK83 334317693 Sinme_2991 Desulfomicrobium baculatum 256828156 Dbac_0341 DSM 4028 Pseudomonas putida W619 170722908 PputW619_3745

Enzymatic Capability 3: Oxidation of Isobutyryl-CoA to Methacrylyl-CoA

A third step in the MMA precursor bioproduction process is the oxidation of isobutyryl-CoA to (methacrylic acid)-coA (MAA-CoA). This step may be performed by a suitable enzyme, such as an isobutryl-coA dehydrogenase enzyme.

The host microorganism may be transformed to express one or more isobutyrylcoA dehydrogenase or equivalent enzymes. For example, the host microorganism may be engineered to express the Pseudomonas aeruginosa ACD1 gene (Genbank Accession Number NP 249437) or an equivalent gene. Other isobutyryl-CoA dehydrogenase enzymes that may be used are listed in Table 6.

TABLE 6 ACD1 Genes Genome Gene ID# Locus Tag Glaciecola nitratireducens 348029414 GNIT_1998 FR1064 Alcanivorax dieselolei B5 407698116 B5T_04301 Gordonia 378719237 GPOL_c37480 polylsoprenlvorans VH2 Pseudogulbenkiania sp. 347540610 NH8B_2828 NH8B Alteromonas macleodii 406596805 MASE_09250 ATCC 27126 Phacobactor 400754588 PGA2_c17140 gallaeciensis 2.10 Xanthomonas oryzae pv. 384420101 XOC_3195 oryzicola BLS256 Mycobacterium 392414871 Mycch_0987 chubuense NBB4 Mycobacterium canettii 340625771 MCAN_07561 CIPT 140010059 Pseudoxanthomonas 357416594 DSC_04600 spadix BD-a59 Mycobacterium 392385470 UDA_0752c tuberculosis UT205 Collimonas fungivorans 340785743 CFU_0549 Ter331 Mycobacterium 385997532 MTCTRI2_0769 tuberculosis CTRI-2 Mycobacterium 379749004 OCU_42850 intracellulare ATCC 13950 Xanthomonas campestris 384427173 XCR_1514 pv. raphani 756C Bordetella pertussis CS 384203847 BPTD_1429 Nocardiopsis alba ATCC 403509890 B005_2435 BAA-2165 Mycobacterium 383306654 MRGA327_04680 tuberculosis RGTB327 Mycobacterium 397678864 MYCMA_0631 massiliense str. GO 06 Rhodospirillum rubrum 386349902 F11_09435 F11 Rhodospirillum 384262441 RSPPHO_02032 photometricum DSM 122 Mycobacterium 386003783 MRGA423_04680 tuberculosis RGTB423 Mycobacterium 397672563 RVBD_0752c tuberculosis H37Rv Mycobacterium bovis 378770509 BCGMEX_0774c BCG str. Mexico Shewanella baltica 386325263 Sbal175_2834 BA175 Streptomyces cattleya 386359035 SCATT_53870 NRRL 8057 = DSM 46488 NC_017586 Bradyrhizobium 384219558 BJ6T_58810 japonicum USDA 6 Brucella melitensis M5-90 384211682 BM590_A1316 Alteromonas macleodii 407683813 AMEC673_09580 str. ‘English Channel 673’ Tistrella mobilis 389877230 TMO_1372 KA081020-065 Roseobacter litoralis Och 339504104 RLO149_c025980 149 Streptomyces 386838951 SHJG_2862 hygroscopicus subsp. jinggangensis 5008 Marinobacter 387813520 MARHY1099 hydrocarbonoclasticus ATCC 49840 Pseudomonas 386064418 NCGM2_1472 aeruginosa NCGM2.S1 Alteromonas macleodii 407687740 AMBAS45_09805 str. ‘Balearic Sea AD45’ Mycobacterium 379756320 OCO_43080 intracellulare MOTT-02 Vibrio furnissii NCTC 375133520 vfu_B01430 11218 Shewanella baltica 386340494 Sbal117_1607 OS117 Mycobacterium rhodesiae 375137842 MycrhN_0640 NBB3 Brucella melitensis NI 384445362 BMNI_I1275 Desulfomonile tiedjei 392411422 Desti_3103 DSM 6799 Mycobacterium africanum 339630821 MAF_07630 GM041182 Brucella canis HSK 376276076 BCA52141_I3086 A52141 Pseudomonas 386060354 PAM18_4293 aeruginosa M18 Xanthomonas 346724209 XACM_1296 axonopodis pv. citrumelo F1 Gordonia sp. KTR9 404216431 KTR9_3836 Shewanella baltica 378707897 Sbal678_1564 OS678 Alteromonas macleodii 407700062 AMBLS11_09065 str. ‘Black Sea 11’ Legionella pneumophila 397666547 LPV_1001 subsp. pneumophila NC_018140 Marinobacter adhaerens 385330648 HP15_907 HP15 Mycobacterium 392431180 TBXG_000759 tuberculosis KZN 605 Stenotrophomonas 344205641 BurJV3_0221 maltophilia JV3 Legionella pneumophila 378776822 lp12_0890 subsp. pneumophila ATCC 43290 Brucella melitensis M28 384408789 BM28_A1325 Bradyrhizobium sp. 383772525 S23_42840 S23321 Mycobacterium 385993820 CCDC5079_0695 tuberculosis CCDC5079 Stenotrophomonas 386716688 SMD_0230 maltophilia D457 Legionella pneumophila 397663427 LPO_0948 subsp. pneumophila NC_018139 Oceanimonas sp. GK1 374336854 GU3_15190 Shewanella putrefaciens 386313180 Sput200_1414 200 Brucella pinnipedialis 340790932 BPI_I1367 B2/94 Kitasatospora setae KM- 357387288 KSE_03230 6054 Mycobacterium 385990221 CCDC5180_0687 tuberculosis CCDC5180 Sinorhizobium fredii 398351439 USDA257_c15560 USDA 257 Phaeobacter 399992913 PGA1_c17340 gallaeciensis DSM 17395 Mycobacterium 375294971 TBSG_00770 tuberculosis KZN 4207 Legionella pneumophila 54293844 lpl0900 str. Lens Aeromonas salmonicida 145298895 ASA_1914 subsp. salmonicida A449 Azorhizobium 158422200 AZC_0576 caulinodans ORS 571 Shewanella woodyi ATCC 170727539 Swoo_3200 51908 Shewanella denitrificans 91793296 Sden_1941 OS217 Rhodomicrobium vannielii 312116097 Rvan_3412 ATCC 17100 Vibrio vulnificus YJ016 37676701 VVA1041 Xanthobacter 154246133 Xaut_2190 autotrophicus Py2 Vibrio sp. Ex25 262396842 VEA_001547 Shewanella oneidensis 24373247 SO_1679 MR-1 Shewanella halifaxensis 167624790 Shal_2876 HAW-EB4 Rhodococcus erythropolis 226307441 RER_39540 PR4 Pseudoalteromonas 109897274 Patl_0949 atlantica T6c Legionella pneumophila 54296890 lpp0931 str. Paris Rhodospirillum centenum 209964651 RC1_1349 SW Pseudomonas 330502345 MDS_1431 mendocina NK-01 Laribacter hongkongensis 226939151 LHK_00217 HLHK9 Bradyrhizobium 27379066 blr3955 japonicum USDA 110 Rhodobacter sphaeroides 146277380 Rsph17025_1335 ATCC 17025 Legionella longbeachae 289164236 LLO_0891 NSW150 Brucella melitensis ATCC 225852804 BMEA_A1361 23457 Chelativorans sp. BNC1 110632946 Meso_0589 Arthrobacter arilaitensis 308178599 AARI_28290 Re117 Vibrio vulnificus CMCP6 27366926 VV2_0492 Aeromonas hydrophila 117620593 AHA_2080 subsp. hydrophila ATCC 7966 Polymorphum gilvum 328543528 SL003B_1909 SL003B-26A1 Shewanella loihica PV-4 127512598 Shew_1669 Mycobacterium 148660528 MRA_0761 tuberculosis H37Ra Shewanella baltica 153000023 Shew185_1491 OS185 Variovorax paradoxus 319793062 Varpa_2389 EPS Microbacterium 323358382 MTES_1934 testaceum StLB037 Vibrio parahaemolyticus 28900477 VPA0622 RIMD 2210633 Xanthomonas campestris 66769286 XC_2980 pv. campestris str. 8004 Rhodopseudomonas 90424584 RPC_3093 palustris BisB18 Rhodospirillum rubrum 83593170 Rru_A1835 ATCC 11170 Mesorhizobium ciceri 319783100 Mesci_3403 biovar biserrulae WSM1271 Ferroglobus placidus 288931943 Ferp_579 DSM 10642 Streptomyces cattleya 357402954 SCAT_5388 NRRL 8057 = DSM 46488 NC_016111 Mycobacterium 253797697 TBMG_00766 tuberculosis KZN 1435 Alicycliphilus denitrificans 319763240 Alide_2557 BC Brucella abortus A13334 376272954 BAA13334_I01881 Alicycliphilus denitrificans 330825434 Alide2_2873 K601 Sphingomonas wittichii 148553574 Swit_0650 RW1 Nakamurella multipartita 258654336 Namu_4214 DSM 44233 Psychrobacter sp. PRwf-1 148652112 PsycPRwf_0300 Pseudoalteromonas sp. 315126581 PSM_A1501 SM9913 Rhodobacter capsulatus 294677101 RCAP_rcc01564 SB 1003 Shewanella sediminis 157374614 Ssed_1475 HAW-EB3 Acidovorax avenae 326317408 Acav_2601 subsp. avenae ATCC 19860 Rhodopseudomonas 39936511 RPA3448 palustris CGA009 Comamonas testosteroni 264678921 CtCNB1_2786 CNB-1 Brucella abortus bv. 1 str. 62290217 BruAb1_1314 9-941 Pseudomonas 392985774 PADK2_21970 aeruginosa DK2 Brevundimonas 302382986 Bresu_1875 subvibrioides ATCC 15264 Sphingobium 334344716 Sphch_1072 chlorophenolicum L-1 Shewanella sp. MR-7 114048154 Shewmr7_2662 Hahella chejuensis KCTC 83643532 HCH_00641 2396 Shewanella violacea 294141872 SVI_3101 DSS12 Mycobacterium bovis 31791939 Mb0774c AF2122/97 Novosphingobium 87198880 Saro_0858 aromaticivorans DSM 12444 Rhodopseudomonas 115524393 RPE_2385 palustris BisA53 Bordetella bronchiseptica 33601607 BB2630 RB50 Nitrobacter winogradskyi 75676343 Nwi_2158 Nb-255 Xanthomonas oryzae pv. 58581466 XOO1843 oryzae KACC10331 Bradyrhizobium sp. BTAi1 148254990 BBta_3579 Nocardia cyriacigeorgica 379707353 NOCYR_1108 GUH-2 Xanthomonas 21242066 XAC1313 axonopodis pv. citri str. 306 Mycobacterium avium 118465062 MAV_4418 104 Shewanella frigidimarina 114562518 Sfri_1340 NCIMB 400 Hirschia baltica ATCC 254295049 Hbal_2701 49814 Mesorhizobium 337268363 Mesop_3886 opportunistum WSM2075 Shewanella baltica 126173735 Sbal_1497 OS155 Stenotrophomonas 190572339 Smlt0265 maltophilia K279a Vibrio harveyi ATCC BAA- 156976709 VIBHAR_05484 1116 Mycobacterium bovis 224989147 JTY_0773 BCG str. Tokyo 172 Bordetella petrii DSM 163856823 Bpet2511 12804 Brucella ovis ATCC 148560349 BOV_1275 25840 Micrococcus luteus 239916835 Mlut_02820 NCTC 2665 Colwellia psychrerythraea 71279027 CPS_0658 34H Pseudovibrio sp. FO- 374331938 PSE_3596 BEG1 Mesorhizobium loti 13471277 mlr1201 MAFF303099 Psychrobacter 93006342 Pcryo_1516 cryohalolentis K5 Rhodobacter sphaeroides 221639593 RSKD131_1494 KD131 Pseudomonas 152984346 PSPA7_4774 aeruginosa PA7 Acidianus hospitalis W1 332795998 Ahos_0309 Acidovorax citrulli AAC00-1 120611270 Aave_2602 15607892 Rv0752c Isosphaera pallida ATCC 320105221 Isop_3707 43644 Maricaulis maris MCS10 114570096 Mmar10_1546 Aeromonas veronii B565 330829883 B565_2183 Xanthomonas campestris 21230718 XCC1261 pv. campestris str. ATCC 33913 Methylobacterium sp. 4- 170740707 M446_2476 46 Dinoroseobacter shibae 159044299 Dshi_1750 DFL 12 Bordetella pertussis 33592543 BP1445 Tohama I Rhodococcus opacus B4 226360658 ROP_12440 Rhodococcus opacus B4 226363908 ROP_44980 Brucella melitensis bv. 1 17986972 BMEI0689 str. 16M Shewanella baltica 160874644 Sbal195_1527 OS195 Acidothermus 117927618 Acel_0409 cellulolyticus 11B Xanthomonas campestris 78046920 XCV1364 pv. vesicatoria str. 85-10 Variovorax paradoxus 239816229 Vapar_3255 S110 Ruegeria pomeroyi DSS-3 56697074 SPO2211 Achromobacter 311106012 AXYL_02830 xylosoxidans A8 Ruegeria sp. TM1040 99080944 TM1040_1103 Beijerinckia indica subsp. 182677610 Bind_0617 Indica ATCC 9039 Nitrobacter hamburgensis 92118071 Nham_2558 X14 Pseudoalteromonas 77360399 PSHAa1456 haloplanktis TAC125 Caulobacter crescentus 221234349 CCNA_01412 NA1000 Stenotrophomonas 194363998 Smal_0220 maltophilia R551-3 Hyphomonas neptunium 114800043 HNE_0900 ATCC 15444 Sphingobium japonicum 294012056 SJA_C1-20700 UT26S Shewanella sp. W3-18-1 120599501 Sputw3181_2702 Nocardia farcinica IFM 54023005 nfa10380 10152 Nocardiopsis dassonvillei 297563334 Ndas_4413 subsp. dassonvillei DSM 43111 Chromobacterium 34497539 CV_2084 violaceum ATCC 12472 Nocardioides sp. JS614 119715626 Noca_1390 Shewanella baltica 217974012 Sbal223_2854 OS223 Vibrio sp. EJY3 375263283 VEJY3_20606 Roseobacter denitrificans 110679512 RD1_2243 OCh 114 Mycobacterium sp. 387877677 W7S_21525 MOTT36Y Acidovorax ebreus TPSY 222111004 Dtpsy_1811 Idlomarina loihiensis 56459977 IL0869 L2TR Caulobacter sp. K31 167647017 Caul_3055 Herbaspirillum 300311865 Hsero_2550 seropedicae SmR1 Caulobacter crescentus 16125599 CC_1350 CB15 Shewanella pealeana 157962602 Spea_2781 ATCC 700345 Mycobacterium sp. 333991932 JDM601_3292 JDM601 Rhodopseudomonas 192292303 Rpal_3937 palustris TIE-1 Syntrophus aciditrophicus 85858067 SYN_02587 SB Rhodococcus equl 103S 312139135 REQ_17170 Mycobacterium 399985881 MSMEI_1461 smegmatis str. MC2 155 NC_018289 Shewanella amazonensis 119774514 Sama_1377 SB2B Brucella abortus S19 189024452 BAbS19_I12450 Magnetospirillum 83312685 amb3586 magneticum AMB-1 Novosphingobium sp. 334141518 PP1Y_AT20310 PP1Y Xanthomonas oryzae pv. 188577295 PXO_01704 oryzae PXO99A Thermobitida tusca YX 72162048 Tfu_1647 Rhodopseudomonas 86749241 RPB_2120 palustris HaA2 Halomonas elongata 307545013 HELO_2423 DSM 2581 Pseudomonas 218893310 PLES_45971 aeruginosa LESB58 Mycobacterium 15840166 MT0776 tuberculosis CDC1551 Bordetella avium 197N 187478253 BAV1758 Parvibaculum 154253338 Plav_2898 lavamentivorans DS-1 Alteromonas macleodii 332141327 MADE_1009645 str. ‘Deep ecotype’ Bordetella parapertussis 33596196 BPP1552 12822 Brucella microti CCM 256369734 BMI_I1326 4915 Gordonia bronchialis 262203762 Gbro_3901 DSM 43247 Sphingopyxis alaskensis 103488529 Sala_3053 RB2256 Mycobacterium 379763856 OCQ_44200 intracellulare MOTT-64 Xanthomonas oryzae pv. 84623396 XOO_1739 oryzae MAFF 311018 Rhodopseudomonas 91977767 RPD_3301 palustris BisB5 Mycobacterium sp. Spyr1 315445940 Mspyr1_44290 Caulobacter segnis ATCC 295689060 Cseg_1650 21756 Shewanella sp. ANA-3 117921210 Shewana3_2769 Shewanella putrefaciens 146292501 Sputcn32_1399 CN-32 Rhodobacter sphaeroides 126462554 Rsph17029_1789 ATCC 17029 Mycobacterium ulcerans 118616613 MUL_0836 Agy99 Mycobacterium sp. MCS 108798036 Mmcs_1063 Mycobacterium marinum M 183981098 MMAR_1078 Catenulispora acidiphila 256393670 Caci_4530 DSM 44928 Mycobacterium bovis 121636675 BCG_0803c BCG str. Pasteur 1173P2 Pseudomonas 116048661 PA14_54630 aeruginosa UCBPP-PA14 Methylobacterium 170750182 Mrad2831_3784 radiotolerans JCM 2831 Acidovorax sp. JS42 121594363 Ajs_2007 Photobacterium 54302789 PBPRB1110 profundum SS9 Phenylobacterium 197105143 PHZ_c1680 zucineum HLK1 Paracoccus denitrificans 119387185 Pden_4480 PD1222 Alteromonas sp. SN2 333893005 ambt_07750 Shewanella sp. MR-4 113970930 Shewmr4_2595 Streptomyces avermitilis 29833461 SAV_6919 MA-4680 Rhodopseudomonas 316933290 Rpdx1_1928 palustris DX-1 Verminephrobacter 121610468 Veis_3538 eiseniae EF01-2 Vibrio vulnificus MO6- 320158804 VVM_01997 24/O Mycobacterium avium 41410312 MAP4214c subsp. paratuberculosis K 10 Brucella melitensis biovar 82700138 BAB1_1333 Abortus 2308 Rhodococcus jostii RHA1 111024023 RHA1_ro07073 Ochrobactrum anthropi 153009201 Oant_1871 ATCC 49188 Erythrobacter litoralis 85374832 ELI_10025 HTCC2594 Actinosynnema mirum 256380353 Amir_6366 DSM 43827 Mycobacterium sp. KMS 119867131 Mkms_1079 Amycolicicoccus 333920326 AS9A_2660 subflavus DQS3-9A1 Mycobacterium 120402372 Mvan_1361 vanbaalenii PYR-1 Legionella pneumophila 148360485 LPC_2425 str. Corby Legionella pneumophila 52841104 lpg0868 subsp. pneumophila str. Philadelphia 1 Acidiphilium multivorum 326403394 ACMV_12460 AIU301 Legionella pneumophila 296106448 lpa_01311 2300/99 Alcoy Mycobacterium gilvum 145225587 Mflv_5011 PYR-GCK Acidiphilium cryptum JF-5 148259637 Acry_0623 Xanthomonas campestris 188992437 xccb100_3042 pv. campestris str. B100 Delftia acidovorans SPH-1 160899128 Daci_3693 Bradyrhizobium sp. 146340123 BRADO3138 ORS278 Psychrobacter arcticus 71065461 Psyc_0901 273-4 Methylobacterium 220922160 Mnod_2176 nodulans ORS 2060 Rhodobacter sphaeroides 77463725 RSP_0156 2.4.1 Carnobacterium sp. 17-4 328958473 CAR_c21860 Alcanivorax borkumensis 110832882 ABO_0021 SK2 Saccharopolyspora 134098043 SACE_1457 erythraea NRRL 2338 Pusillimonas sp. T7-7 332284306 PT7_1053 Shewanella piezotolerans 212636148 swp_3385 WP3 Tsukamurella 296140757 Tpau_3069 paurometabola DSM 20162 Streptomyces griseus 182435009 SGR_1216 subsp. griseus NBRC 13350 Mycobacterium 118472185 MSMEG_1497 smegmatis str. MC2 155 NC_008596 Jannaschia sp. CCS1 89054400 Jann_1909 Mycobacterium 169628281 MAB_1188c abscessus ATCC 19977 Mycobacterium 148821957 TBFG_10766 tuberculosis F11 Glaciecola sp. 4H-3-7 + YE5 332307811 Glaag_3460 Marinobacter aquaeolei 120555047 Maqu_2132 VT8 Mycobacterium sp. JLS 126433697 Mjls_1090 Pseudomonas 146306407 Pmen_1375 mendocina ymp Ferrimonas balearica 308048737 Fbal_1020 DSM 9799 Sinorhizobium meliloti 334320231 sinme_4289 AK83

Enzymatic Capability 4: From 3-Hydroxyisobutyryl-CoA to MAA-CoA

In the bioproduction of 3-HIB from valine, one step is the conversion of MAA-CoA to 3-hydroxyisobutyryl-CoA. This may be accomplished by a suitable enzyme, such as an enoyl-CoA hydratase.

The host microorganism may be transformed to express one or more enoyl-coA hydratase (ECH) or equivalent enzymes. For example, the host microorganism may be engineered to express the Pseudomonas aeruginosa echA gene (Genbank Accession Number NP 249436) or an equivalent gene. Alternatively, an ECH gene selected from Table 7 may be used.

Enzymatic Capability 5: From 3-Hydroxyisobutyryl-CoA to 3-HIB

In the bioproduction of 3-HIB from valine, the final enzymatic step is the conversion of 3-hydroxyisobutyryl-CoA to 3-HIB by cleaving of the CoA from 3-hydroxyisobutyryl. This may be accomplished by a suitable enzyme, such as a thioesterase.

The host microorganism may be transformed to express one or more enzymes that creave CoA from 3-hydroxyisobutyryl-CoA. For example, the host microorganism may be transformed to express one or more thioesterase enzymes that cleave CoA from 3-hydroxyisobutyryl-CoA (HCH genes). For example, the host microorganism may be engineered to express the Pseudomonas aeruginosa hchA gene (Genbank Accession Number NP 249435) or an equivalent gene. Alternatively, an HCH gene selected from Table 7 may be used.

TABLE 7 Genome Gene ID# Locus Tag Desulfosporosinus 374994422 Desor_1769 orientis DSM 765 Pseudogulbenkiania sp. 347540611 NH8B_2829 NH8B Methylophaga sp. 387130393 Q7C_1447 JAM7 Xanthomonas oryzae 384420100 XOC_3194 pv. oryzicola BLS256 Pseudoxanthomonas 357416595 DSC_04605 spadix BD-a59 Pseudomonas stutzeri 339493199 PSTAB_1122 ATCC 17588 = LMG 11199 Pseudomonas stutzeri 386019805 PSTAA_1177 DSM 4166 Pyrobaculum sp. 1860 374326631 P186_1146 Collimonas fungivorans 340785744 CFU_0550 Ter331 Bacillus megaterium 384044695 BMWSH_0519 WSH-002 Xanthomonas 384427174 XCR_1515 campestris pv. raphani 756C Amycolatopsis 384149061 RAM_19670 mediterranei S699 NC_017186 Leptospira interrogans 386073441 LIF_A0972 serovar Lai str. IPAV Turneriella parva DSM 392405679 Turpa_4152 21527 Shewanella baltica 386325262 Sbal175_2833 BA175 Ornithobacterium 392390035 Ornrh_0644 rhinotracheale DSM 15997 Marinobacter 387813521 MARHY1100 hydrocarbonoclasticus ATCC 49840 Pseudomonas 386064417 NCGM2_1471 aeruginosa NCGM2.S1 Vibrio furnissii NCTC 375133521 vfu_B01431 11218 Burkholderia sp. KJ006 387903700 MYA_2947 Shewanella baltica 386340495 Sbal117_1608 OS117 Pseudomonas 386060355 PAM18_4294 aeruginosa M18 Xanthomonas 346724210 XACM_1297 axonopodis pv. citrumelo F1 Pseudomonas stutzeri 397686059 PSJM300_04720 DSM 10701 Amycolatopsis 399537636 AMES_3817 mediterranei S699 V2 NC_018266 Shewanella baltica 378707898 Sbal678_1565 OS678 Flavobacterium indicum 383449484 KQS_00745 GPTSA100-9 Marinobacter 385330649 HP15_908 adhaerens HP15 Burkholderia cepacia 402568213 GEM_3472 GG4 Stenotrophomonas 344205642 BurJV3_0222 maltophilia JV3 Pseudomonas stutzeri 392420207 A458_05710 CCUG 29243 Bacillus cereus F837/76 376267399 bcf_17380 Stenotrophomonas 386716689 SMD_0231 maltophilia D457 Burkholderia 386864395 BP1026B_110685 pseudomallei 1026b Owenweeksia 375013016 Oweho_2388 hongkongensis DSM 17368 Leptospirillum 383785540 LFE_2308 ferrooxidans C2-3 Oceanimonas sp. GK1 374336853 GU3_15185 Shewanella 386313181 Sput200_1415 putrefaciens 200 Aeromonas salmonicida 145298896 ASA_1915 subsp. salmonicida A449 Shewanella woodyi 170727538 Swoo_3199 ATCC 51908 Shewanella 91793295 Sden_1940 denitrificans OS217 Vibrio vulnificus YJ016 37676700 VVA1040 Vibrio sp. Ex25 262395672 VEA_000372 Shewanella oneidensis 24373248 SO_1680 MR-1 Shewanella halifaxensis 167624789 Shal_2875 HAW-EB4 Planctomyces 325107014 Plabr_0433 brasiliensis DSM 5305 Sorangium cellulosum 162454201 sce5924 ‘So ce 56’ Cupriavidus 194291860 RALTA_B1107 taiwanensis LMG 19424 Pseudomonas 330502344 MDS_1430 mendocina NK-01 Laribacter 226939152 LHK_00218 hongkongensis HLHK9 Croceibacter atlanticus 298207322 CA2559_03685 HTCC2559 Sideroxydans 291613755 Slit_1288 lithotrophicus ES-1 Pedobacter saltans 325105493 Pedsa_2784 DSM 12145 Vibrio vulnificus 27366925 VV2_0491 CMCP6 Aeromonas hydrophila 117621061 AHA_2081 subsp. hydrophila ATCC 7966 Thioalkalivibrio 220933275 Tgr7_0083 sulfidophilus HL-EbGr7 Shewanella loihica PV-4 127512599 Shew_1670 Shewanella baltica 153000024 Shew185_1492 OS185 Pseudomonas stutzeri 146281598 PST_1214 A1501 Ralstonia pickettii 12D 241665652 Rpic12D_4090 Vibrio parahaemolyticus 28900478 VPA0623 RIMD 2210633 Xanthomonas 66769285 XC_2979 campestris pv. campestris str. 8004 Xylella fastidiosa 28198324 PD0407 Temecula1 Burkholderia 126457284 BURPS1106A_A0830 pseudomallei 1106a Leptospira interrogans 45658337 LIC12495 serovar Copenhageni str. Fiocruz L1-130 Polaromonas sp. JS666 91788338 Bpro_2473 Bacillus cereus E33L 52142006 BCZK3237 Geobacillus 138895542 GTNG_1892 thermodenitrificans NG80-2 Burkholderia glumae 238024069 bglu_2g06300 BGR1 Bacillus thuringiensis 118478779 BALH_3171 str. AI Hakam Fluviicola taffensis DSM 327403570 Fluta_1578 16823 Pseudomonas fulva 12-X 333901441 Psefu_3258 Burkholderia 126443955 BURPS668_A0922 pseudomallei 668 Burkholderia gladioli 330820009 bgla_2g08970 BSR3 Azotobacter vinelandii 226943481 Avin_13550 DJ Psychrobacter sp. 148652111 PsycPRwf_0299 PRwf-1 Pseudoalteromonas sp. 315126582 PSM_A1502 SM9913 Ralstonia eutropha 73538090 Reut_B4260 JMP134 Shewanella sediminis 157374615 Ssed_1476 HAW-EB3 Pseudomonas 392985775 PADK2_21975 aeruginosa DK2 Xylella fastidiosa M12 170729668 Xfasm12_0460 Frankla alnl ACN14a 111219689 FRAAL0193 Burkholderia phymatum 186473921 Bphy_5130 STM815 Shewanella sp. MR-7 114048153 Shewmr7_2661 Hahella chejuensis 83643531 HCH_00640 KCTC 2396 Shewanella violacea 294141871 SVI_3100 DSS12 Burkholderia sp. 323528140 BC1001_3821 CCGE1001 Robiginitalea biformata 260061101 RB2501_05870 HTCC2501 Xanthomonas oryzae 58581467 XOO1844 pv. oryzae KACC10331 Thiobacillus 74318690 Tbd_2672 denitrificans ATCC 25259 Xanthomonas 21242067 XAC1314 axonopodis pv. citri str. 306 Burkholderia ambifaria 172063912 BamMC406_4900 MC40-6 Burkholderia 76818705 BURPS1710b_A2182 pseudomallei 1710b Shewanella 114562519 Sfri_1341 frigidimarina NCIMB400 Shewanella baltica 126173736 Sbal_1498 OS155 Stenotrophomonas 190572340 Smlt0266 maltophilia K279a Vibrio harveyi ATCC 156976710 VIBHAR_05485 BAA-1116 Ruminococcus albus 7 317056589 Rumal_1928 Burkholderia 91780315 Bxe_C0248 xenovorans LB400 Burkholderia 189353613 BMULJ_04857 multivorans ATCC 17616 NC_010801 Colwellia 71277850 CPS_0657 psychrerythraea 34H Maribacter sp. 305665647 FB2170_05090 HTCC2170 Psychrobacter 93006341 Pcryo_1515 cryohalolentis K5 Ralstonia eutropha H16 116695134 H16_B1189 Pseudomonas 152986979 PSPA7_4775 aeruginosa PA7 Bacillus atrophaeus 311067529 BATR1942_02825 1942 Aeromonas veronii 330829882 B565_2182 B565 Xanthomonas 21230719 XCC1262 campestris pv. campestris str. ATCC 33913 Burkholderia 206564029 BCAM2192 cenocepacia J2315 Shewanella baltica 160874645 Sbal195_1528 OS195 Xanthomonas 78046921 XCV1365 campestris pv. vesicatoria str. 85-10 Burkholderia sp. 383 78061568 Bcep18194_B0718 Bacillus pseudofirmus 288555720 BpOF4_13560 OF4 Burkholderia 161520209 Bmul_3660 multivorans ATCC 17616 NC_010084 Sulfurovum sp. NBC37-1 152992154 SUN_0558 Polynucleobacter 171463376 Pnec_0628 necessarius subsp. necessarius STIR1 Pseudoalteromonas 77360400 PSHAa1457 haloplanktis TAC125 Xylella fastidiosa subsp. 386084484 XFLM_07430 fastidiosa GB514 Stenotrophomonas 194363999 Smal_0221 maltophilia R551-3 Shewanella sp. W3-18-1 120599500 Sputw3181_2701 Chromobacterium 34497538 CV_2083 violaceum ATCC 12472 Shewanella baltica 217974011 Sbal223_2853 OS223 Vibrio sp. EJY3 375262877 VEJY3_18566 Bacillus megaterium 294501468 BMQ_4732 QM B1551 Herbaspirillum 300311864 Hsero_2549 seropedicae SmR1 Shewanella pealeana 157962601 Spea_2780 ATCC 700345 Bacillus cereus 225865490 BCA_3601 03BB102 Ralstonia pickettii 12J 187926164 Rpic_3977 Burkholderia 134292051 Bcep1808_3333 vietnamiensis G4 Mycobacterium 399988423 MSMEI_4020 smegmatis str. MC2 155 NC_018289 Shewanella 119774515 Sama_1378 amazonensis SB2B Bacillus cellulosilyticus 317129856 Bcell_3165 DSM 2522 Xanthomonas oryzae 188577294 PXO_01705 pv. oryzae PXO99A Burkholderia 83717948 BTH_II1799 thailandensis E264 Pseudomonas 218893311 PLES_45981 aeruginosa LESB58 Xanthomonas oryzae 84623397 XOO_1740 pv. oryzae MAFF 311018 Burkholderia 187920332 Bphyt_5646 phytofirmans PsJN Shewanella sp. ANA-3 117921209 Shewana3_2768 Shewanella 146292502 Sputcn32_1400 putrefaciens CN-32 Burkholderia 53721657 BPSS0621 pseudomallei K96243 Cupriavidus necator N-1 339322459 CNE_2c11550 Burkholderia 107025765 Bcen_3408 cenocepacia AU 1054 Pseudomonas 116048660 PA14_54640 aeruginosa UCBPP- PA14 Bacillus megaterium 295706816 BMD_4718 DSM 319 Photobacterium 54302788 PBPRB1109 profundum SS9 Burkholderia 170737684 Bcenmc03_5327 cenocepacia MC0-3 Burkholderia sp. 307727573 BC1003_5577 CCGE1003 Burkholderia 116693053 Bcen2424_4959 cenocepacia HI2424 Xylella fastidiosa M23 182680963 XfasM23_0401 Shewanella sp. MR-4 113970929 Shewmr4_2594 Amycolatopsis 300785752 AMED_3862 mediterranei U32 Vibrio vulnificus MO6- 320158803 VVM_01994 24/O Sulfurimonas 307721221 Saut_1301 autotrophica DSM 16294 Acaryochloris marina 158334786 AM1_1621 MBIC11017 Leptospira interrogans 24213898 LA_1198 serovar Lai str. 56601 Leptospira biflexa 189910448 LBF_0894 serovar Patoc strain ‘Patoc 1 (Ames)’ Methylotenera sp. 301 297539640 M301_2470 Burkholderia ambifaria 115359129 Bamb_4381 AMMD Lysinibacills 169828468 Bsph_2967 sphaericus C3-41 Burkholderia sp. 295699817 BC1002_4206 CCGE1002 Xanthomonas 188992436 xccb100_3041 campestris pv. campestris str. B100 Psychrobacter arcticus 71065462 Psyc_0902 273-4 Xylella fastidiosa 9a5c 15837717 XF1115 Shewanella 212636147 swp_3384 piezotolerans WP3 Mycobacterium 118468149 MSMEG_4119 smegmatis str. MC2 155 NC_008596 Brevibacillus brevis 226311256 BBR47_16690 NBRC 100599 Herpetosiphon 159897731 Haur_1202 aurantiacus ATCC 23779 Leptospira biflexa 183220331 LEPBI_I0927 serovar Patoc strain ‘Patoc 1 (Paris)’ Marinobacter aquaeolei 120555046 Maqu_2131 VT8 Pseudomonas 146306406 Pmen_1374 mendocina ymp Fenimonas balearica 308048738 Fbal_1021 DSM 9799 Sphingobacterium sp. 326798186 Sph21_0758 21

Enzymatic Capability 6: From MAA-CoA to MMA

Enzymatic Capability 6 comprises the ability to convert MAA-CoA to MMA by cleavage of the CoA from MAA-CoA. Such activity may be imparted by an alcohol acyl transferase (AAT) enzyme. In one embodiment, the AAT enzyme is coded by the Malus pumila AAT gene. In one embodiment, the host microorganism is yeast. In another embodiment, the host microorganism is yeast and the AAT gene is a Malus pumila AAT gene codon-optimized for expression in yeast, for example the AAT gene comprising SEQ ID No: 2. Alternatively, an AAT gene selected from Table 9 may be used.

TABLE 8 AAT Homologs and Orthologs. Gene Gene Descriptor Gene Descriptor (Uniprot ID number) (Uniprot ID number) A0A0B2SBV5 Q64FJ6 I1J859 V9P9R1 UPI0003D6F583 UPI000498BC7F UPI0003D788E3 V9P9L8 V4TGK2 Q6QLX5 A8W8Y0 UPI000498A175 W9STV1 V9P9M2 W9S561 U5GMN1 A0A068BGA5 A0A067K2U6 UPI00051082F8 UPI0005FC091A UPI000511B89C Q8GV03 UPI000498D560 UPI0005811CF4 A9YCD1 M5W9C4 U5GQY0 M5WLR7 UPI000579EB90 UPI00046DB41D UPI00051192DA K4BYU6 Q5GJ80 UPI000523FDB9 UPI00051161D8 A0A059C378 UPI0004988A4D UPI0005205BA1 UPI000511AD74 M5WU76 UPI00049877DB UPI000498EB94 UPI000498867B UPI00057A1605 Q6R311 UPI00057A4ABE V9QNV9 B9NG88 UPI000498C0D7 UPI000499245B A0A0B4VC61 B3VP15 V9P9T8 M1C8D7 V4W0X4 UPI0003D7778A UPI00046DDD34

Enzymatic Capabilities

In the invention, examples of the origin of the above enzymes (genes encoding enzymes) include genus Pseudomonas, genus Bacillus, genus Sphingobacterium, genus Comamonas, genus Brevundimonas, genus Sphingomonas, genus Ochrobactrum, genus Pedobacter, genus Paenibacillus, genus Achromobacter, genus Acinetobacter, genus Shewanella, genus Listonella, genus Agrobacterium, genus Mesorhizobium, genus Rhizobium, genus Paracoccus, genus Xanthobacter, genus Streptomyces, genus Geobacillus, genus Rhodococcus, genus Saccharomyces, genus Candida or genus Aspergillus. Of these, genus Pseudomonas and genus Rhodococcus microorganisms are preferable.

Examples of the microorganism classified into genus Pseudomonas include Pseudomonas aeruginosa, Pseudomonas agarici, Pseudomonas alcaligenes, Pseudomonas amygdale, Pseudomonas anguiliseptica, Pseudomonas antimicrobica, Pseudomonas aspleni, Pseudomonas aurantiaca, Pseudomonas aureofaciens, Pseudomonas avellanae, Pseudomonas azotoformans, Pseudomonas balearica, Pseudomonas beijerinckii, Pseudomonas beteli, Pseudomonas boreopolis, Pseudomonas carboxyhydrogena, Pseudomonas caricapapayae, Pseudomonas cichorii, Pseudomonas cissicola, Pseudomonas citronellolis, Pseudomonas coronafaciens, Pseudomonas corrugate, Pseudomonas doudoroffii, Pseudomonas echinoids, Pseudomonas elongate, Pseudomonas ficuserectae, Pseudomonas flavescens, Pseudomonas flectens, Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas fulva, Pseudomonas fuscovaginae, Pseudomonas gelidicola, Pseudomonas geniculata, Pseudomonas glathei, Pseudomonas halophila, Pseudomonas hibiscicola, Pseudomonas huttiensis, Pseudomonas iners, Pseudomonas lancelota, Pseudomonas lemoignei, Pseudomonas lundensis, Pseudomonas luteola, Pseudomonas marginalis, Pseudomonas meliae, Pseudomonas mendocina, Pseudomonas mucidolens, Pseudomonas monteilli, Pseudomonas nautica, Pseudomonas nitroreducens, Pseudomonas oleovorans, Pseudomonas oryzihabitans, Pseudomonas pertucinogena, Pseudomonas phenazinium, Pseudomonas pictorum, Pseudomonas pseudoalcaligenes, Pseudomonas putida, Pseudomonas pyrrocinia, Pseudomonas resinovorans, Pseudomonas rhodesiae, Pseudomonas saccharophila, Pseudomonas savastanoi, Pseudomonas spinosa, Pseudomonas stanieri, Pseudomonas straminae, Pseudomonas stutzeri, Pseudomonas synxantha, Pseudomonas syringae, Pseudomonas syzygii, Pseudomonas taetrolens, Pseudomonas tolaasii, Pseudomonas veronii, Pseudomonas viridiflava, Pseudomonas vulgaris and Pseudomonas wisconsinensis.

Examples of the microorganism classified into genus Rhodococcus include Rhodococcus rhodochrous, Rhodococcus erythropolis, Rhodococcus equi, Rhodococcus opacus, Rhodococcus jostii, Rhodococcus pyridinovorans, Rhodococcus rhodnii, Rhodococcus corallinus, Rhodococcus rubropertinctus, Rhodococcus coprophilus, Rhodococcus globerulus, Rhodococcus chlorophenolicus, Rhodococcus luteus, Rhodococcus aichiensis, Rhodococcus chubuensis, Rhodococcus maris and Rhodococcus fascians.

For the genes encoding the enzyme, RNA is extracted from the above microorganism cell in accordance with a routine method, a primer is designed based on amino acid sequences and gene sequences of the above enzymes (the microorganism or related species thereto) disclosed in the public databases such as NCBI, and PCR can be carried out using the primer to thereby isolate and amplify the gene encoding the enzyme of interest.

Methacrylyl-CoA-Producing Microorganism

The invention encompasses an engineered microorganism capable of producing MAA-CoA from valine. Such an engineered microorganism of the invention comprises the microorganism which possesses Enzymatic Capability 1, Enzymatic Capability 2, and Enzymatic Capability 3, as described above.

The enzyme may be produced in the cytosol or may be targeted to any other cellular compartment. In one embodiment, the enzyme introduced by transformation and expressed by the MAA-CoA producing microorganism is targeted to the mitochondria. In one embodiment, the mitochondrial-targeted enzyme is targeted to the mitochondrial matrix. In one embodiment, the mitochondrial-targeted protein comprises a targeting moiety comprising the Su9 or Cox1 presequence. In one embodiment, the engineered microorganism is a yeast. In one embodiment, the yeast is Saccharomyces cerevisiae.

In one embodiment, the engineered microorganism has been transformed to express one or more acyl-CoA dehydrogenases (isobutyryl-CoA dehydrogenase). For example, the MAA-CoA producing microorganism is transformed to express the Pseudomonas aeruginosa ACD1 gene. In another embodiment, the MAA-coA producing microorganism is transformed to express one or more enzymes which catalyze the decarboxylation of 2-oxoisovalerate to isobutyryl-CoA. For example, the MAA-CoA producing microorganism is transformed to express one, some, or all of the components of the BCKAD complex. In yet another embodiment, the MAA-CoA producing microorganism of the invention is transformed to express one or more enzymes which catalyze the formation of 2-oxoisovalerate from valine, for example the BCAT1 or BCAT2 genes from yeast.

3-HIB-Producing Microorganism

The scope of the invention encompasses host microorganisms which have been engineered to produce (S)-3-hydroxyisobutyric acid (3-HIB) from valine. 3-HIB is an MMA precursor which can be readily converted to MMA utilizing various methods known in the art.

The 3-HIB-producing microorganism of the invention is a microorganism which possesses Enzymatic Capability 1, Enzymatic Capability 2, Enzymatic Capability 3, Enzymatic Capability 4, and Enzymatic Capability 5.

In one embodiment, the 3-HIB-producing microorganism of the invention is a host cell that has been transformed to express an ECH gene, for example, the echA gene from Pseudomonas aeruginosa. In another embodiment, the 3-HIB-producing microorganism of the invention is a host cell that has been transformed to express one or more HCH enzymes that cleave CoA from 3-hydroxyisobutyryl-CoA. For example the 3-HIB producing microorganism may comprise an microorganism which expresses the hchA gene from Pseudomonas aeruginosa. In one embodiment, the engineered microorganism is a yeast. In one embodiment, the yeast is Saccharomyces cerevisiae. In one embodiment, the enzyme introduced by transformation and expressed by MAA-CoA producing microorganism is targeted to the mitochondria. In one embodiment, the mitochondrial-targeted enzyme is targeted to the mitochondrial matrix. In one embodiment, the mitochondrial-targeted protein comprises a targeting moiety comprising the Su9 or Cox1 matrix-targeting presequence.

MMA-Producing Microorganism

In one embodiment, the engineered microorganism of the invention is capable of producing MMA. An MMA-producing microorganisms of the invention comprises an microorganism which possesses Enzymatic Capability 1, Enzymatic Capability 2, Enzymatic Capability 3, and Enzymatic Capability 6. In one embodiment, the MMA-producing microorganism of the invention has been transformed to express an alcohol acyl transferase (AAT), for example the AAT gene of Malus pumila. In one embodiment, the AAT gene is encoded by SEQ 1D NO: 2. In one embodiment, the MMA-producing microorganism is a yeast. In one embodiment, the yeast is Saccharomyces cerevisiae. In one embodiment, the mitochondrial-targeted enzyme is targeted to the mitochondrial matrix. In one embodiment, the mitochondrial-targeted protein comprises a targeting moiety comprising the Su9 presequence.

Additional Genetic Modification

In addition to the various combinations of Enzymatic Capabilities described above, the engineered microorganisms of the invention may further comprise genetic modifications to promote MMA end-product formation. For example, the starting material for bioproduction of MMA end-product is valine. Accordingly, in one embodiment, the microorganisms of the invention are engineered to express one or more enzymes which enhance valine formation over wild-type levels. Various genetic modifications are known in the art for enhancing valine formation in various microorganisms, for example as described in Literature [Wada et al., 2008, Enhanced Valine Production in Corynebacterium glutamicum with Defective H+-ATPase and C-Terminal Truncated Acetohydroxyacid Synthase, Bioscience, Biotechnology, and Biochemistry 72 (11):2959-65, 2008; Hasegawa et al., 2013, Engineering of Corynebacterium glutamicum for high-yield L-valine production under oxygen deprivation conditions, Appl Environ Microbiol, 79(4):1250-7; and Park et al., 2007, Metabolic engineering of Escherichia coli for the production of L-valine based on transcriptome analysis and in silico gene knockout simulation, PNAS 104 (19) 7799-7802]. The enzyme or regulatory gene inserted to a microorganism to enhance the formation of valine may be under the control of a constitutive promoter or inducible promoter.

Under some culture conditions, such as highly aerated yeast cultures, biosynthetic pathways, such as Erlich pathway reactions, compete with MMA end-product formation. In some embodiments, the engineered microorganism of the invention is further engineered to downregulate or obliterate competing biosynthetic pathway. For example, in one embodiment, the bioengineered microorganisms of the invention is engineered to express miRNA or other sequences that downregulate or otherwise target enzymes which compete with any of Enzymatic Capabilities 1 to 6. For example, in one embodiment, the microorganism of the invention is engineered with an enzymatic or a regulatory gene which reduces Erlich pathway reaction. In another embodiment, the enzymatic or regulatory gene co-introduced into the host species comprise genes which inhibit valine catabolism, for example, the reaction which forms succinyl Co-A from 3-HIB Co-A. Such gene may be placed under the control of an inducible promoter, allowing downregulation to be induced after cultures have been propagated and are being switched to bioproduction mode.

In another embodiment, the engineered microorganism of the invention is further engineered to express an enzyme or a regulatory sequence which enhances the regeneration of co-factor such as NADH or FADH. For example, in one embodiment, the engineered microorganism of the invention is transformed to express a high level of alcohol dehydrogenase, in order to promote NADH formation from NADPH, which such cofactor is necessary for the action of the BCKAD complex. Likewise, genetic modifications may be introduced that increase the rate of regeneration of FAD from FADH2, a cofactor in the action of ACD. Exemplary cofactor manipulation technologies are described in Literatures [Wang et al., “Engineering of cofactor regeneration enhances (2S,3S)-2,3-butanediol production from diacetyl,” Sci Rep. 2013; 3:2643. doi: 10.1 038/srep02643; Nikel et al., “Elimination of D-lactate synthesis increases poly(3-hydroxybutyrate) and ethanol synthesis from glycerol and affects cofactor distribution in recombinant Escherichia coli,” Appl Environ Microbiol. 2010 November; 76 (22):7400-6; Tseng and Prather, “Controlled biosynthesis of odd-chain fuels and chemicals via engineered modular metabolic pathways,” Proc Natl Acad Sci USA. 2012 Oct. 30; 109(44):17925-30; and Lopez de Felipe et al., “Cofactor Engineering: a Novel Approach to Metabolic Engineering in Lactococcus/actis by Controlled Expression of NADH Oxidase,” J Bacteriol. 1998 August; 180(15): 3804-3808].

Methacrylic Acid Ester and Methacrylic Acid Ester Precursor Production

The scope of the invention encompasses the engineered microorganism described above, and further encompasses a method of using such an engineered microorganism to produce the end-products such as MMA.

The engineered microorganisms are cultured so as to propagate themselves and to produce the methacrylic acid esters or methacrylic acid ester precursors end-products. The engineered microorganisms may be cultured under continuous culture conditions for the sustained growth of cultures and simultaneous harvest of end-products. Alternatively, the microorganisms may be cultured in batches wherein discreet cultures (e.g. in a single vessel or bioreactor) are used to form end products, and then are processed to recover the end-products.

In some embodiments, the culture of the engineered microorganisms is staged, with a first growth and/or propagation stage followed by a bioproduction stage. The various stages may be implemented by altering culture conditions, wherein the initial growth and propagation stage is fostered by the use of media and/or culture conditions that favor rapid propagation and growth of the engineered microorganism, followed by a change in culture conditions to favor MMA end-product formation.

For example, in one embodiment, the engineered microorganism of the invention is a yeast and is first grown under culture conditions that favor rapid propagation and growth of yeast cultures, for example well aerated conditions with high levels of nutrients in the growth media. This growth stage is followed by a bioproduction stage wherein the cultures are not highly aerated or are not aerated at all and the culture medium lacks one or more nutrients (e.g. micronutrients, or carbon sources that promote growth).

Each condition such as culture temperature or culture time is suitably determined without particular limitation depending on raw materials, microorganisms to be used, and end-products of interest, but the reaction may be usually carried out at 5 to 80° C. for 1 minute to 1 week. The reaction is preferably carried out at 10 to 70° C. for 1 minute to 120 hours, with 10 minutes or more being more preferable. The conditions under which the reaction quenches are preferably selected from these conditions. The pH of the reaction solution is also not particularly limited as long as the reaction proceeds effectively and, for example, pH ranges from 4 to 10, with pH 5.5 to 8.5 being preferable.

For the purpose of effectively progressing the reaction, the culture can also be carried out in a system to which an organic solvent is added in advance. For the organic solvent, for example, linear, branched or cyclic saturated or unsaturated aliphatic hydrocarbons, or saturated or unsaturated aromatic hydrocarbons can be used singly or in combinations of two or more. Specific examples include hydrocarbon solvents (e.g., pentane, hexane, cyclohexane, benzene, toluene and xylene), halogenated hydrocarbon solvents (e.g., methylene chlorides and chloroform), ether solvents (e.g., diethyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, t-butyl methyl ether and dimethoxyethane) and ester solvents (e.g., methyl formate, methyl acetate, ethyl acetate, butyl acetate and methyl propionate).

In one embodiment, following the growth and propagation stage, the cells are collected and placed in culture vessels having conditions favoring higher bioproduction. For example, yeast cells may be isolated from growth and propagation cultures by centrifugation, followed by rinsing in buffer and resuspension in new culture medium.

In microorganisms wherein one or more of the proteins comprising the Enzymatic Capabilities is under the control of an inducible promoter, the inducing agent may be introduced near or at the beginning of the bioproduction stage. Likewise, in microorganisms which have been engineered for decreased or inhibited activity of metabolic pathways that compete with or otherwise reduce MMA end-product formation, and where such introduced genes are under the control of inducible promoters. The cultured cells may be exposed to the inducing agent at the transition from the growth and propagation stage to the bioproduction stage.

In some embodiments, valine is added to the culture medium to enhance formation of MMA end-products from valine. In another embodiment, valine is added to the culture medium at the transition to a bioproduction culture stage. In another embodiment, valine precursors are included in the growth media to improve valine formation and drive increased rates of MMA end-product formation. Likewise, in microorganisms which have been engineered for increased valine formation by the introduction of genes and where such genes are under the control of inducible promoters, the inducing agent may be introduced at or near the beginning of the bioproduciton stage to increase the amount of precursors for end-product formation. In some embodiments, the engineered microorganisms of the invention are co-cultured with other strains or microorganisms which produce valine, in order to increase the concentration of valine in the culture medium.

During culture, in the case of continuous production systems, or at the completion of the bioproduction stage, in the case of batch cultures, the end-products are recovered from the culture. This may be accomplished by any means. For example in one embodiment, enzymatic and/or physical treatments are applied to lyse cells and to liberate the end-products. The MMA end-products may be isolated from cell cultures by centrifugation, distillation, column separation, chromatography, and other means known in the art. For example, methodologies described in U.S. Pat. No. 8,907,121, by Johnson and Morris, entitled “Methyl methacrylate purification process,” may be employed.

In the case of methacrylic acid ester precursors, the precursor recovered from cultures may be further treated to convert them to a methacrylic acid esters. 3-HIB may be treated enzymatically to convert it to MMA. For example, the dehydration of 3-HIB to MMA may be achieved, for example as described in Literature [Nagai, “New Developments in the Production of Methyl Methacrylate,” Applied Catalysis A: General 221 (2001) 367-377].

The method of the invention can also be used for the production of methacrylic acid esters or precursors thereof from a biomass. A transformant having introduced thereinto the gene encoding an acyl-CoA dehydratase, as well as an enzyme gene group allowing for synthesizing methacrylic acid esters or precursors thereof of interest from a biomass can be used to directly synthesize methacrylic acids or methacrylic acid esters from the biomass in a metabolic engineering (fermentation) approach.

The additional details in regard to the experiment methods described in the invention and Example 1 are presented in FIGS. 2 and 5.

Example Mitochondrial Expression of Pathway Genes

Genes were targeted for expression to the cytoplasm by expressing the heterologous gene, or targeted to the mitochondria by appending the first 69 residues of subunit 9 of the yeast mitochondrial ATPase (Su9) to each gene as encoded by SEQ ID NO: 1.

Expression and correct targeting of these genes was then validated by constructing a C-terminal GFP fusion which was expressed from a GAL1 promoter within the plasmid pYES (leader peptide, plasmid, GAL1 promoter, CYCT terminator and GFP, all provided from the paper: Westermann, B. and Neupert, W. (2000). Yeast 16: 1421-1427). These constructs were transformed in Saccharomyces cerevisiae (CKY263) and grown overnight in glucose supplemented synthetic defined media lacking uracil (SD-CAA). At 24 hour, the cultures were diluted 100 fold and expression induced by growing them in galactose supplemented SO-CAA for 24 hours. The cells were harvested and stained with a red fluorescent mitochondria-selective dye (Mito-IO Red detection kit-Cat# ENZ-51007-500, Enzo Life Sciences, Ann Arbor, Mich.). Fluorescence was then detected using confocal microscopy-only cells with non-diffuse green fluorescence which overlaps with the red dye target gene expression to the mitochondria.

Recombinant Activity of Targeted MMA Pathway Genes

Activity of the first two enzymatic steps (BCKAO and ACO) were confirmed with in vitro assays of crude lysates from cells that expressed GFP-free constructs. MMA enzymes expression cassettes (BCKAO, ACO, ECH, HCH) targeted to either the mitochondria (mtMMA) or cytoplasm (MMA) were cloned from the previous experiment into the pRS series of vectors (pBCKA04-pRS315 backbone (LEU) with bkdA1, bkdA2, bkdB, JpdV; pAC01-pRS316 backbone (URA) with acd1; and pCoA2-pRS314 backbone (TRP) with echA and hchA). These plasmids were transformed into the vacuolar protease-deficient Saccharomyces cerevisiae strain BJ5464. After a 48-hour induction period in galactose supplemented SO-CAA lacking uracil, leucine and tryptophan, cells were harvested, physically disrupted in Tris buffer and the lysate separated by centrifugation. The lysates were then assayed with real-time enzymatic activity assays as depicted below:

ACD Assay

100 mM Potassium phosphate pH 8.0

1.0 mM N-Ethylmaleimide 0.03 mM Isobutyryl-CoA

0.4 mM Flavin adenine dinucleotide (FAD) 1.6 mM Phenazine methosulphate (PMS)

0.035 mM 2,6-Dichlorophenol-indophenol (DCPIP) +Sample

Room temperature incubation Active ACD reduces DCPIP and A₆₀₀

BCKAD Assay

100 mM Potassium phosphate pH 7.0

1 mM MgCl₂

0.2 mM Thiamin pyrophosphate (TPP) 4 mM 2-Oxoisovaleric acid

0.2 mM CoASH/2 mM DTT 2 mM NAD⁺ 2 mM L-valine +Sample

Room temperature incubation Active BCKAD reduces NAD⁺ and increases A₃₄₀

Production of 3-Hydroxyisobutyrate

Productivity from the (mt)MMA pathway was assessed using high-pressure liquid chromatography. BJ5464 cells containing mitochondrial or cytoplasmic variants of the pathway were grown overnight under non-inducing conditions. These cultures were then diluted about 100 fold in inducing SD-CAA media and grown for 48 to 72 hours. Supernatant from these cultures were then analyzed on an Agilent 1100 series HPLC with an ICsep USP L-17 using a 0.01 N H₂SO₄ mobile phase. 3-HIB product was detected at 210 nm with a DAD detector and quantified via a standard curve. These studies suggest production of g/L titers of 3-HIB (5.1±2.9 g/L) in 72 hours with the mitochondrial pathway variant.

All patents, patent applications, and publications cited in this specification are herein incorporated by reference in their entirety to the same extent as if each independent patent, patent application, or publication was specifically and individually indicated to be incorporated by reference. The disclosed embodiments are presented for purposes of illustration and not limitation. While the invention has been described with reference to the described embodiments thereof, it will be appreciated by those of skill in the art that modifications can be made to the structure and elements of the invention without departing from the spirit and scope of the invention as a whole.

INDUSTRIAL APPLICABILITY

The invention is useful for synthesizing methacrylic acid esters such as MMA to begin with and acrylic resins, which are polymers thereof. 

1. A eukaryotic microorganism into which a gene encoding an acyl-CoA dehydrogenase (isobutyryl-CoA dehydrogenase) is introduced.
 2. The eukaryotic microorganism according to claim 1, wherein the gene encoding an acyl-CoA dehydrogenase is derived from at least one selected from genus Pseudomonas, genus Bacillus, genus Sphingobacterium, genus Comamonas, genus Brevundimonas, genus Sphingomonas, genus Ochrobactrum, genus Pedobacter, genus Paenibacillus, genus Achromobacter, genus Acinetobacter, genus Shewanella, genus Listonella, genus Agrobacterium, genus Mesorhizobium, genus Rhizobium, genus Paracoccus, genus Xanthobacter, genus Streptomyces, genus Geobacillus, genus Rhodococcus, genus Saccharomyces, genus Candida and genus Aspergillus.
 3. The eukaryotic microorganism according to claim 1, wherein a signal sequence-added acyl-CoA dehydrogenase gene is introduced in such a way that the acyl-CoA dehydrogenase expresses a function in a mitochondrion.
 4. The eukaryotic microorganism according to claim 3, wherein the signal sequence comprises the sequence as set forth in SEQ ID NO:
 1. 5. The eukaryotic microorganism according to claim 1, wherein the eukaryotic microorganism is a yeast.
 6. The eukaryotic microorganism according to claim 1, further comprising at least one exogenous gene selected from genes encoding branched-chain keto acid dehydrogenase, genes encoding enoyl-CoA hydratase, genes encoding hydroxyacyl-CoA hydrolase, genes encoding thioesterase, and genes encoding alcohol acyl transferase.
 7. A method for producing methacrylyl-CoA from valine using a eukaryotic microorganism according to claim
 6. 8. A method for producing 3-hydroxyisobutyryl-CoA from valine using a eukaryotic microorganism according to claim
 6. 9. A method for producing 3-hydroxyisobutyric acid from valine using a eukaryotic microorganism according to claim
 6. 10. A method for producing a methacrylic acid ester from valine using a eukaryotic microorganism according to claim
 6. 